WO2005023985A2 - Cytidine deaminase activators, deoxycytidine deaminase activators, vif antagonists, and methods of screening for molecules thereof - Google Patents

Cytidine deaminase activators, deoxycytidine deaminase activators, vif antagonists, and methods of screening for molecules thereof Download PDF

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WO2005023985A2
WO2005023985A2 PCT/US2004/028796 US2004028796W WO2005023985A2 WO 2005023985 A2 WO2005023985 A2 WO 2005023985A2 US 2004028796 W US2004028796 W US 2004028796W WO 2005023985 A2 WO2005023985 A2 WO 2005023985A2
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vif
ceml
cytidine deaminase
rna
apobec
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PCT/US2004/028796
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French (fr)
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WO2005023985A3 (en
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Harold C. Smith
Joseph E. Wedekind
Mark P. Sowden
Stephen Dewhurst
Baek Kim
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University Of Rochester
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Publication of WO2005023985A3 publication Critical patent/WO2005023985A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04005—Cytidine deaminase (3.5.4.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04014—Deoxycytidine deaminase (3.5.4.14)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • HIV- 1 a human lentivirus, is the causative agent of AIDS, which presently infects approximately 42 million persons worldwide with 1 million infected persons in North America (http://www.unaids.org). The high mutation rate of HIV- 1 has in the past made it impossible to develop therapies that retain their effectiveness. Cunent therapies for HIV infected patients target the production of new viras by antiviral agents
  • Human white blood cells express a protein called CEM15, a cytidine deaminase, which can change the genetic code of the infecting AEDS viruses.
  • Vif interrupting deaminase functions in other systems such as the apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1) and Activation Induced Deaminase (AED) systems have similar significance in the treatment of other diseases such as hypercholesterolemia and Hyper-IgM syndrome and certain forms of cancer (i.e., colorectal, APOBEC-1 and various leukemias and lymphomas).
  • APOBEC-1 apolipoprotein B mRNA editing catalytic subunit 1
  • AED Activation Induced Deaminase
  • this invention in one aspect, relates to Vif antagonists,.
  • This invention also relates to cytidine deaminase activators, CEM15 activators, APOBEC-1 activators, and AED activators, and methods of identifying and making such agents.
  • this invention relates to deoxycytidine deaminase activators, ARP activators, and methods of identifying and making such activators.
  • Figure 1 shows the effect of introns on editing efficiency.
  • A Diagram of the chimeric apoB expression constructs. The intron sequence (EVS) is derived from the adeno viras late leader sequence. Co-ordinates of the human apoB sequence are shown and the location of PCR amplimers indicated. X indicates the deleted 5' splice donor or 3' splice acceptor sequences. CMV is cytomegalo virus.
  • EVS intron sequence
  • X indicates the deleted 5' splice donor or 3' splice acceptor sequences.
  • CMV is cytomegalo virus.
  • B Poisoned-primer-extension Attorney Docket Number 21108.0035P1
  • FIG. 8 shows the effect of intron proximity on editing efficiency.
  • A Diagram of the chimeric apoB expression constructs. EVS-(EVS ⁇ 3'5')-apoB and EVS- (IVS ⁇ 3'5') -apoB were created by the insertion of one or two copies respectively of the EVS ⁇ 3'5' intron cassette into EVS-apoB. Human apoB co-ordinates and amplimer annealing sites are indicated ( Figure 1).
  • B Poisoned-primer-extension assays of amplified apoB RNAs. Pre-mRNA and mRNA were amplified with the MS7/MS2 or SP6/T7 amplimers respectively. Editing efficiencies, an average for duplicate transfections, for each RNA are shown beneath. 9. Figure 3 shows that the editing sites within introns are poorly utilized.
  • A Diagram of the chimeric apoB expression constructs. EVS-(EVS ⁇ 3'5')-apoB and EVS- (IVS ⁇ 3'5')
  • FIG. 10 shows that editing is regulated by RNA splicing.
  • A Diagram of the modified CAT reporter construct (CMV128) used in the Rev complementation assay; a gift from Dr Thomas J. Hope of the Salk Institute. The splice donor (SD), splice acceptor (SA), RRE, intron and 3' long tandem repeat (LTR) are from the HEV-1 genome. CMV128 was modified by insertion of the apoB editing cassette as a PCR product into the BamHI site 3' of the CAT gene. Amplimer annealing sites are indicated.
  • B McArdle cell CAT activity in the absence (Vector) or presence of the Rev transactivator. Values are averages for duplicate experiments. CMVCAT was an assay confrol transfection.
  • C Poisoned-primer-extension assays of amplified apoB Attorney Docket Number 2110S.0035P1
  • RNAs 'hitron and exon RNA' was amplified using the EF MS2 amplimers. Editing efficiencies for each RNA are shown beneath. Promiscuous editing is indicated by ' 1 '. 11.
  • Figure 5 shows representative members of the APOBEC-1 related family of cytidine deaminases including CEM15. Also are APOBEC-1 complementation factor (ACF) and viral infectivity factor (Vif).
  • ACF APOBEC-1 complementation factor
  • Vif viral infectivity factor
  • the catalytic domain of APOBEC-1 is characterized by a ZDD with three zinc ligands (either His or Cys), a glutamic acid, a proline residue and a conserved primary sequence spacing (Mian, I.S., et al, (1998) J Comput Biol.
  • LRR forms the hydrophobic core of the protein monomer
  • ACF complements APOBEC-1 through its APOBEC-1 and RNA bindings activities.
  • the RNA recognition motifs (RRM)s are required for mooring sequence-specific RNA binding and these domains plus sequence flanking them are required for APOBEC-1 interaction and complementation (Blanc, V., et al, (2001) J Biol Chem. 276:46386-93.; Mehta, A., et al., (2002) RNA.
  • APOBEC-1 complementation activity minimally depends on ACF binding to both APOBEC-1 and mooring sequence RNA.
  • a broad APOBEC-1 complementation region is indicated that is inclusive of all regions implicated in this activity (Blanc, V., et al, (2001) J Biol Chem. 276:46386-93.; Mehta, A., et al, (2002) RNA. 8:69-82.)
  • Experiments have shown the N-terminal half of Vif is necessary for viral infectivity (Henzler, T. 2001). However, reports have demonstrated that residues in the C-terminus (amino acids 151-164) are essential for infectivity (Yang, S. et al.
  • Figure 6 shows schematic depictions of the cytidine deaminase (CD A) polypeptide fold and structure-based alignments of APOBEC-1 with respect to its related proteins (ARPs).
  • Figure 6a depicts a gene duplication model for cytidine deaminases.
  • CDD1 belongs to the teframeric class of cytidine deaminases with a quaternary fold nearly identical to that of the teframeric cytidine deaminase from B. subtilis (Johansson, E., et al, (2002) Biochemistry. 41:2563-70.).
  • Such teframeric enzymes exhibit the classical ⁇ topology of the Zinc Dependent Deaminase Domain (ZDD) observed first in the Catalytic Domain (CD) of the dimeric enzyme from E. coli (Betts, L., et al, (1994) J Mol Biol. 235:635-56).
  • ZDD Zinc Dependent Deaminase Domain
  • CD Catalytic Domain
  • an ancesfral CDDl-like monomer (upper left ribbon) duplicated and fused to produce a bipartite monomer.
  • PCD C-terminal Pseudo-Catalytic Domain
  • the model holds that the interdomain CD-PCD junction is joined via flexible linker that features conserved Gly residues necessary for catalytic activity on large polymeric DNA or RNA substrates.
  • the function of the PCD is to stabilize the hydrophobic monomer core and to engage in auxiliary factor binding.
  • the loss of PCD helix ⁇ l can provide a hydrophobic surface were auxiliary factors bind to facilitate substrate recognition thereby regulating catalysis.
  • the enzymes remain oligomeric because each active site comprises multiple polypeptide chains. Modern representatives of the chimeric CDA fold include the enzyme from E. coli, as well as APOBEC-1 and AED.
  • ARPs such as APOBEC-3G (CEM15) may have arisen through a second gene duplication to produce a pseudo-homodimer on a single polypeptide chain (lower ribbon); structural properties of the connector polypeptide are unknown.
  • Signature sequences compiled from strict structure-based alignments (upper) are shown below respective ribbon diagrams, where X represents any amino acid. Linker regions (lines) and the location of Zn 2+ binding (spheres) are depicted.
  • FIG. 6b depicts the stracture based sequence alignment for ARPs. Sequences from human APOBEC-1, 5 AED, and APOBEC-3G were aligned based upon a main-chain alpha-carbon least- squares superposition of the known cytidine deaminase three dimensional crystal stractures from E. coli, B. subtilis and S. cerevisiae ( Figure 6c). Amino acid sequence alignments were optimized to minimize gaps in major secondary stracture elements, which are depicted as tubes ( ⁇ -helices) and anows ( ⁇ -strands) in Figure 6b.
  • L-Cl and L-C2 represent distinct loop structures in the dimeric versus teframeric cytidine deaminases. Sections of basic residues that overlap the bipartite NLS of APOBEC-1 are marked BP-1 and BP-2.
  • Figure 6d depicts a schematic diagram of the domain structure observed in APOBEC-1 and related ARPs based upon
  • Figure 7 shows the relation of CEM15 amino acid sequence to APOBEC-1 and other APOBEC-1 Related Proteins (ARPs) by use of standard computational methods based upon amino acid similarity or identity. Amino acid sequence alignments
  • ZDD zinc-dependent deaminase domain
  • CSR Switch Recombination
  • CEM15 which blocks HIV-1 viral infectivity
  • Figure 8 shows a schematic ribbon diagram depicting a three-dimensional model of APOBEC-1 derived from comparative modeling by the method of satisfying spatial restraints. Structure-based homology modeling has provided insight into the fold of APOBEC-1, and has been conoborated by protein engineering, site-directed
  • the cunent model for APOBEC-1 predicts a two domain stracture comprising a catalytic domain (CD) and a pseudo-catalytic domain (PCD) joined by a central linker, which folds over the active site (green segment).
  • CD catalytic domain
  • PCD pseudo-catalytic domain
  • linker sequence is conserved among ARPs ( Figure 6b), and linker sequence composition and polypeptide chain length are essential for efficient RNA editing by APOBEC-1.
  • the APOBEC-1 model also provides a rationale for losses in editing due to surface point mutations, such as F156L, located 25 A from the active site. This 5 aromatic to branched-chain hydrophobic change appears to have no influence on the stability of the enzyme core, but can be involved in auxiliary factor binding required for RNA binding.
  • a series of basic residues at BP2 ( Figure 6b) are close to the active site, and can be responsible for RNA binding. Mutagenesis of all basic residues within the respective bp-clusters abolishes editing activity (Teng, 1999, J. Lipid Res.
  • the stractural template of the APOBEC-1 model is derived from the spatial constraints derived from a superposition of three high resolution CDA crystal stractures that exhibit a nearly identical ⁇ 2 ⁇ fold despite modest sequence identity ( ⁇ 24% Figure 6c); fold conservation also exists at the oligomeric level, since each enzyme exhibits either proper 222 or pseudo ⁇ 222 symmetry. Similarities in the Zn 2+ dependent
  • CEM15 (APOBEC-3G) evolved from an APOBEC-1 -like precursor by gene duplication.
  • the CEM15 stracture comprises two active sites per polypeptide chain with the
  • Figure 9 shows a structural model for CEM15.
  • the use of comparative modeling by the method of satisfied spatial restraints has allowed the calculation of a CEM15 three-dimensional model including all atoms of the 384 amino sequence. Spatial restraints for the template were derived from the atomic coordinates of three
  • CDA crystal structures including a bonafi.de RNA editing enzyme from yeast Cddl, which is capable of deaminating free nucleosides as well as polymeric RNA substrates, such as reporter apoB mRNA.
  • the known CDA crystal structures represent both dimeric and teframeric quaternary folds ( Figure 6a), which allows an accurate model to be prepared using multiple structural restraints. Further insight into the
  • CEM15 stracture has also been attained by analogy to modeling and functional results obtained from APOBEC-1.
  • a comparative model of CEM15 was calculated by use of the program 'Modeller' and subsequently checked by the program suites PROCHECK Attorney Docket Number 21108.0035P 1
  • the model was energy minimized using simulated annealing and molecular dynamics methods. No restraints were placed on secondary elements, except those derived from the triple CDA stracture alignment. The position of the UMP nucleotide was incorporated based upon spatial restraints derived from known 5 crystal structures. Zn atoms were restrained using reasonable coordination geometry derived from the known CD As. The resulting model demonstrated that the 384 amino acid sequence of CEM15 can be accommodated by a dimeric CDA quaternary fold (analogous to the E. coli CDA or APOBEC-1 with 2 x 236 amino acids).
  • Figure 10 shows possible CEM15 oligomers. The number of possible
  • CEM15 quaternary stractures is limited and the actual oligomeric state can be evaluated by gel filtration chromatography, or through site directed mutagenesis that evaluates the requirement of single or dual CD domains in CEM15 activity.
  • possible dimeric CEM15 stractures Figures 10c and lOd predict mutually exclusive intermolecular contacts with the distinguishing feature that the interaction observed in
  • Figure 10c is such that each CD pairs with itself, and similarly for each PCD. hi contrast, every domain in Figure lOd falls in a unique environment (i.e. no CD or PCD pairs with itself).
  • a variety of truncation mutations address the question of whether or not a dimer of the form in head-to-head or head-to-tail exists in solution ( Figure 10c versus lOd).
  • Figure 11 shows HA-tagged CEM15 in 293T cells. Stable, HA-tagged
  • CEM15 expressing 293T cell lines were selected with puromycin and verified by western blotting with a HA specific monoclonal antibody. The addition of the HA epitope tag has no effect on the ability of CEM15 to suppress infectivity. Isogenic HEV- 1 pro-viral DNAs are packaged into pseudotyped lentiviral particles by co-transfection
  • Figure 12 shows the results of the assay described in Example 4, indicating that the expression of CEM15 in 293T cells resulted in at least a 100-fold decrease in Vif- viral infectivity compared to particles generated in parental 293T cells. The low
  • Figure 13 shows poisoned primer extension assays and Western analysis for Cddl mutants and chimeric proteins.
  • overexpressed Cddl is capable of C to U specific editing of reporter apoB mRNA at site C 6666 at a level of 6.7%, which is ⁇ 10x times greater than the negative control (Figure 13, empty vector - compare lanes 1 and 2).
  • Figure 13, empty vector - compare lanes 1 and 2 In confrast, the CDA from E. coli (equivalent to PDB entry 1AF2) is incapable of editing on the reporter substrate ( Figure 13, lane 3).
  • the active site mutants E61A and G137A abolish detectable Cddl activity ( Figure 13, lanes 4 and 5).
  • FIG. 21 shows CEM15 suppresses HIV-1 protein abundance.
  • 293 T cell lines stably expressing (A) CEM15, (B) DM, and (C) control pLRES-P vector were transiently fransfected with proviral HEV-1 plasmids (containing either wild-type Vif (+) or ⁇ Vif (-)).
  • Total cell lysates were prepared at 24, 48, and 72 hours post-transfection, separated by SDS-PAGE and analyzed by immunoblot assay using antibodies reactive with HA (HA-tagged CEM15 and DM), Vif, p24, RT, ⁇ - Attorney Docket Number 21108.0035P1
  • actin, Vpr, or Tat (as denoted on the left).
  • the molecular weight (kDa) of the indicated protein species is given to the right.
  • FIG. 16 shows CEM15 suppresses HEV-l viral RNA abundance.
  • A Location of Gag-Pol junction and protease region of HEV-l genomic RNA conesponding to the GP-RNA probe used for RNA binding and northern blot analysis.
  • B UV crosslinking of increasing concentration of recombinant CEM15 protein (1, 2 and 4 ⁇ g protein) to 20 frnol radiolabeled GP-RNA and apoB RNA.
  • C Poly A+ RNA abundance for Gag-Pol transcripts in 293T-CEM15 at 24, 48, and 72 hours and DM cells at 48 hours post- transfection with Vif+ (black) and ⁇ Vif (white) proviral DNA. Results are expressed as the ratio of viral RNA (GP-RNA region) to endogenous cellular RNA (adeno virus El A) determined through phosphorimager scanning densitometry analysis of northern blots.
  • the invention provides compounds that enhance RNA or DNA editing, as well as methods of using, identifying, and making such compounds.
  • the compounds are useful in preventing or treating a variety of diseases, including viral infections. Described herein are cytosine deaminase activators and antagonists of compounds, like viral infectivity factor (vif), that interfere with deaminases.
  • RNA and DNA editing 24 There are several examples of cellular and viral mRNA editing in mammalian cells. (Grosjean and Benne (1998); Smith et al. (1997) RNA 3: 1105-23). Two examples of such editing mechanisms are the adenosine to inosine and cytidine to uridine conversions.
  • Editing can also occur on both RNA and on DNA, and typically these functions are performed by different types of deaminases.
  • a to I editing involves a family of adenosine deaminases active on RNA (ADARs).
  • ADARs typically have two or more double stranded RNA binding motifs (DRBM) in addition to a catalytic domain whose tertiary structure positions a histidine and two cysteines for zinc ion coordination and a glutamic acid residue as a proton donor.
  • DRBM double stranded RNA binding motifs
  • the catalytic domain is conserved at the level of secondary and tertiary structure among ADARs, cytidine nucleoside/nucleotide deaminases and CDARs but Attorney Docket Number 21108.0035P 1
  • ADAR editing sites are found predominantly in exons and are characterized by RNA secondary structure encompassing the adenosine(s) to be edited.
  • RNA secondary structure is formed between the exon and a 3 ' proximal sequence with the downstream infron
  • ADAR mRNA substrates frequently contain multiple A to I editing sites and each site is selectively edited by an ADAR, such as ADAR1 or ADAR2.
  • ADARs typically function autonomously in editing mRNAs. ADARs bind secondary stracture at the editing site through their double stranded RNA binding motifs or DRBMs and perform hydrolytic deamination of adenosine through their catalytic domain.
  • APOBEC-1 26 One example of a Cytosine Deaminase Active on RNA (CDAR) is APOBEC-1 (apolipoprotein B mRNA editing catalytic subunit 1) (accession # NM_005889) encoded on human chromosome 12. (Grosjean and Benne (1998); Lau et al. (1994) PNAS 91:8522-26; Teng et al (1993) Science 260:1816-19).
  • APOBEC- 1 edits apoB mRNA primarily at nucleotide 6666 (C6666) and to a lesser extent at C8702 (Powell et al. (1987) Cell 50:831-40; Chen et al.
  • apoB mRNA editing results in increased production and secretion of B48 containing very low density lipoproteins and conespondingly, a decrease in the abundance of the atherogenic apoBlOO containing low density lipoproteins in serum (Davidson et al. (1988) JBC 262:13482-85; Baum et al. (1990) JBC 265:19263-70; Wu et al. (1990) JBC 265:12312-12316; Harris and Attorney Docket Number 21108.0035P1
  • ApoB is translated from a 14 kb mRNA that is transcribed from a single copy gene located on human chromosome 2 (Scott (1989) J. Mol. Med. 6:65-80).
  • ApoB protein is a non-exchangeable structural component of chylomicrons and of very low density (VLDL) and low density (LDL)
  • APOBEC-1 editing of apoB mRNA determines whether a small (apoB48) or a large (apoBlOO) variant of apoB lipoprotein is expressed (Grosjean and Benne (1998); Powell et al. (1987) Cell 50:831-840; Chen et al. (1987) Science 238:363-66; Scott (1989) J. Mol. Med. 6:63-80; Greeve et al (1993) J. Lipid Res. 34:1367-83).
  • RNA secondary structure does not appear to be required for apoB RNA editing. Instead, apoB mRNA editing requires an 11 nucleotide motif known as the mooring sequence. Placement of the mooring sequence 4-8 nucleotides 3' of a cytidine within reporter RNAs is frequently sufficient for that RNA to support editing (Smith (1993) Seminars in Cell Biol. 4:267-78; Sowden et al.
  • 30 cytidine is predictive of an editing site.
  • APOBEC-1 relies on auxiliary proteins for RNA recognition (Grosjean and Benne (1998); Teng et al. (1993) Science 260:1816-19; Sowden et al (1998) Attorney Docket Number 21108.0035P1
  • APOBEC-1 only has weak RNA binding activity of low specificity (Anant et al. (1995) JBC 270:14768-75; MacGinnitie et al. (1995) JBC 270:14768-75).
  • APOBEC-1 requires a mooring 5 sequence-specific, RNA binding protein that binds apoB mRNA and to which
  • APOBEC-1 can bind and orient itself to C6666.
  • apoB RNA, recombinant APOBEC-1 and proteins known as ACF/ASP (APOBEC-1 Complementing Factor/APOBEC-1 Stimulating Protein) were all that was required for editing activity and are therefore considered as the minimal editing complex or
  • ACF was isolated and cloned using biochemical fractionation and yeast two hybrid genetic selection (Mehta et al. (2000) Mol. Cell Biol. 20:1846-54; Lellek et al. (2000) JBC 275:19848-56).
  • APOBEC-1 (Figure 8; Wedekind et al. Trends Genet, 19(4):207-16, 2003), and the modeling of APOBEC-1 has been conoborated by protein engineering, site-directed Attorney Docket Number 21108.0035P1
  • the cunent model for APOBEC-1 is a two domain structure comprising a catalytic domain (CD) and a pseudo-catalytic domain (PCD) joined by a central linker, which folds over the active site ( Figure 8).
  • the linker sequence is conserved among ARPs, and sequence identity and length are 5 essential for efficient RNA editing by APOBEC- 1.
  • the APOBEC- 1 model also provides a rationale for losses in editing due to surface point mutations, such as F156L (Navaratnam et al. Cell 81(2)187-95), located 25 A from the active site. Such a change can influence auxiliary factor binding.
  • Other mutations such as K33A K34A abolish activity (Teng et al. J Lipid Res, 40(4) 623-35, 1999). 10 32.
  • Other mutations such as K33A/K34A abolish activity (Teng et al. J Lipid
  • dimerization has been shown to be essential for editing activity (Lau et al. (1994) PNAS 91:8522-26; Navaratnam et al. (1995) Cell 81:187-95; Oka et al. (1997) JBC 272:1456-60).
  • the model also predicted a leucine-rich region (LRR) in the C-terminus of APOBEC-1 as a functional motif characteristic of cytidine deaminases that function as dimers.
  • LRR leucine-rich region
  • the LRR is essential for APOBEC-1 homodimer formation, apoB mRNA editing, APOBEC-1 interaction with ACF, and APOBEC-1 subcellular distribution (Lau et al.
  • CDARs have attracted interest because they share homology with the catalytic domain found in APOBEC-1 and the ADARs and they also have interesting physiological circumstances for their expression.
  • One characteristic of the catalytic domain in CDARs and ADARs is the occunence and spacing of a histidine and two cysteines (or three cysteines), required for the Attorney Docket Number 21108.0035P1
  • ZBD zinc binding domain
  • AID contains a ZDD (Zinc-dependent deaminase domain) and has 34% amino acid identity to APOBEC-1 (Table 4, Figure 5 and 6). Its location on human chromosome 12pl3 suggests it may be related to APOBEC-1 by a gene duplication event (Lau, 1994; Muto, 2000). This chromosomal region has been implicated in the autosomal recessive form of Hyper-IgM syndrome (HIGM2) (Revy, 2000). Most patients with this disorder have homozygous point mutations or deletions in three of the five coding exons, leading to missense or nonsense mutations (Revy, P., 2000) Cell. 102:565-75). Significantly, some patients had missense mutations for key amino acids within AED's ZDD (Revy, 2000; Minegishi, 2000).
  • AED homologous knockout mice demonstrated that AED expression was the rate limiting step for class switch recombination (CSR) and required for an appropriate level of somatic hypermutation SHM (Muramatsu, 2000).
  • CSR class switch recombination
  • SHM somatic hypermutation
  • the expression of AED controls antibody diversity through multiple gene reanangements involving mutation of DNA sequence and recombination.
  • the initial expression of antibodies requires immunoglobulin (Ig) gene reanangement that is AED-independent (Muramatsu, M., et al., (2000) Cell 102:553-63). This occurs in immature B lymphocytes developing in fetal liver or adult bone manow and requires DNA double strand breaks at the Ig heavy chain locus whose ends are rejoined by non-homologous end joining.
  • the reananged immunoglobulin V (variable), D (diversity) and J (joining) gene segments encode a variable region that is expressed initially with the
  • mu ( ⁇ ) constant region (C ⁇ ) to form a primary antibody repertoire composed of IgM antibodies.
  • AED-dependent gene alterations occur in B lymphocytes that are growing in germinal centers of secondary lymphoid organs following antigen activation. This involves multiple mutations of the variable region through Somatic Hypermutation (SHM) as well as removing the C ⁇ and replacing it with one of several other constant regions (Ca, Cd, Ce or Cg) through a recombination process known as Class Switch Recombination, CSR.
  • SHM Somatic Hypermutation
  • GC gene conversion
  • AED cannot substitute for APOBEC-1 in the editing of apoB mRNA (Muramatsu, 1999) and, although this negative result may have been expected (given that most editing enzymes have substrate specificity (Grosjean and Benne (1998)), it did suggest that AED may have another activity.
  • a competing hypothesis for AED's role in CSR and SHM is that it deaminates deoxycytidine in DNA (Rada, C. et al.
  • AID overexpression in NTH 3T3 fibroblasts resulted in the deamination of deoxycytidine in DNA encoding a green fluorescent protein (GFP) (Yoshikawa, 2002) and also in antibiotic resistance and metabolic genes when AED expression in bacteria was placed under selection for a mutator' phenotype (Harris, 2002).
  • GFP green fluorescent protein
  • a variety of mutations were observed on GFP DNA including deletions and duplications; however, a preference for transitions at G/C base pairs clustered within regions predicted to have DNA secondary structure was observed. Similar mutations were observed in the bacteria overexpressing AED and their frequency was markedly enhanced when evaluated in an ung-1 background (lacking functional uracil-DNA glycosylase, an enzyme involved in repairing C to T mutations). (Harris, 2002).
  • the target hotspot for AED is characterized by the motif RGYW (R is A or G, Y is C or T and W is A or T) (Honjo et al. Annu Rev Immunol 20:165-96, 2002; Martin et al. Nat
  • dC is deaminated to dU in the sfrand of DNA that is displaced by transcription of RNA (the non-templating sfrand); conoborating other studies in which AID selectively deaminated dC in ssDNA or mutated dsDNA reporters within a nine base pair mismatch (the size of a transcription bubble) (Bransteitter et al. Proc Natl Acad Sci U S A 100(7) :4102-7, 2003; Ramiro et al. Nat Immunol, 2003).
  • AED appears to act processively on DNA, binding initially to RGYW and mutating dC to dU and then modifying multiple dC residues from that point along the same sfrand of DNA.
  • AID's ability to act on DNA would not negate the possibility that it also acts on RNA. Whether AID is involved in DNA and/or RNA modification, its function clearly results in the diversification of expressed genomic sequences.
  • Human APOBEC-2 (Genbank Accession # XM004087) is encoded on chromosome 6 and is expressed uniquely in cardiac and skeletal muscle (Liao et al. Biochem Biophys. Res. Commun. 260:398-404). It shares homology with APOBEC- I's catalytic domain, has a leucine/isoleucine-rich C-terminus and a tandem structural homology of the ZBD in its C-terminus. APOBEC-2 deaminated free nucleotides in vitro but did not have editing activity on apoB mRNA.
  • Phorbolins 1, 2, 3, and Phorbolin-1 related protein were identified in skin from patients suffering from psoriasis and were shown to be induced (in the case of Phorbolins 1 and 2) in skin treated with phorbol 12-myristate-l -acetate (Muramatsu, M. et al. (1999) J Biol Chem. 274:18470-6). The genes for these proteins were subsequently renamed as members of the APOBEC-3 or ARCD family locus (Table 1) (Madsen, P. et al. (1999) J Invest Dermatol. 113:162-9). Bioinfo ⁇ natic studies revealed the presence of two additional APOBEC-1 related proteins in the human genome. One is an expressed gene (XM_092919) located just 2 kb away from APOBEC-3 G, and is thus likely to be an eighth member of the family. The other is at position 12q23, and has similarity to APOBEC-3 G.
  • APOBEC-3 variants show homology to cytidine deaminases (Figure 6d). As anticipated from the SBSA, some of these proteins bind zinc and have RNA binding capacities similar to APOBEC-1 Jarmuz, A., et al, (2002) Genomics, 79:285-96).However, analysis of APOBEC-3A, -3B and -3G revealed them unable to edit apoB mRNA Jarmuz, A., et al, (2002) Genomics, 79:285-96); Muramatsu, M. et al. (1999) J Biol Chem. 274:18470-6).
  • APOBEC-3E appears to be a pseudogene (Jarmuz, A., et al, (2002) Genomics, 79:285-96), yet the EST database shows that APOBEC-3D and APOBEC-3E are alternatively spliced to fonn a single CD-PCD-CD-PCD encoding transcript. Additionally, it has been shown that rat APOBEC- 1 , mouse APOBEC-3 , and human APOBEC-3B, are able to inhibit HEV infectivity even in the presence of Vif. Like APOBEC-3G, human APOBEC-3F preferentially restrict vif-deficient virus.
  • APOBEC-3F The mutation spectra and expression profile of APOBEC-3F indicate that this enzyme, together with APOBEC- 3G, accounts for the G to A hypermutation of pro viruses described in HEV-infected individuals (Bishop et al., Cun. Bio. 14:1392-1396, 2004). In accordance with this, it has also been shown that APOBEC-3F blocks HEV-l and is suppressed by both the HEV-l and HEV-2 Vif proteins (Zheng et al, J Virol 78(11): 6073-6076, 2004; Wiegand et al, EMBO 23:2451-58, 2004).
  • APOBEC-3G (CEM15) has also been shown to interfere with other retroelements, including but not limited to hepatitis B virus (HBV) and murine leukemia virus (MLV).
  • HBV hepatitis B virus
  • MLV murine leukemia virus
  • the methods and compositions described herein are useful with any of these viruses (Bishop et al., Cun. Bio. 14:1392-1396, 2004; Machida et al, PNAS 101(12):4262-67, 2004; Turelli et al., Science, 303:1829, 2004).
  • Table 1 shows APOBEC-1 and related proteins have been described previously (Anant, S., et al., Am J Physiol Cell Physiol. 281:C1904-16.; Dance, G.S., et al, (2001) Nucleic Acids Res. 29:1772-80.; Jarmuz, A., et al., (2002) Genomics. 79:285-96) and extended through amino acid similarity searches with the (1) hidden Markov modeling software SAM trained with CDDl, APOBEC-1, APOBEC-2, AED and Phorbolin 1, (2) PHI-BLAST, using the target patterns H(V/A)-E-X-X-F-X 19 - Attorney Docket Number 21108.0035P1
  • HsARP human APOBEC-1 Related Proteins
  • Human HEV-l virus contains a 10-kb single-stranded, positive-sense RNA genome that encodes three major classes of gene products that include: (i) stractural proteins such as Gag, Pol and Env; (ii) essential trans-acting proteins (Tat, Rev); and
  • Vif auxiliary proteins that are not required for efficient virus replication in at least some cell culture systems.
  • Vif is required for efficient viras replication in vivo, as well as in certain host cell types in vitro (Fisher et al. Science 237(4817):888-93, 1987; Strebel et al. Nature 328(6132):728-30, 1987) because of its ability to overcome the action of a cellular 10 antiviral system (Madani et al. J Virol 72(12):10251-5, 1998; Simon et al. Nat Med 4(12): 1397-400, 1998).
  • vif- ⁇ eleted molecular clones of HEV-l is strikingly different in vz/-permissive cells (e.g. 293T, SUPT1 and CEM-SS T cell lines), as compared to vz -non-permissive cells (e.g. primary T cells,
  • CEM15 antiviral activity is derived from effects on viral RNA or reverse transcripts (Sheehy, A.M., et al., (2002)
  • CEM15 deaminates dC to dU as the first sfrand of DNA is being made by reverse transcriptase or soon after its completion, and this results in dG to dA changes at the conesponding positions during second strand DNA synthesis (Harris et al. Cell 113:803-809, 2003).
  • CEMl 5 The premise of molecular modeling is that primary sequence analysis alone is insufficient to evaluate effectively the anti-viral activity of CEMl 5.
  • the use of comparative modeling of CEMl 5 is based on three known CDA crystal structures (Betts et al. J Mol Biol 235(2):635-56, 1994; Johansson et al. Biochemistry 41(8): p. 2563-70, 2000) and knowledge gained from similar work with APOBEC-1.
  • CEMl 5 modeling has been accomplished by aligning its amino acid sequence onto a composite three-dimensional template derived by superposition (Winn et al. J Synchrotron Radiat, (2003) 10(Pt l):23-5; Kabsch et al. Acta.
  • Crystallogr. (1976) A32:922-923; Potterton et al. Acta Crystallogr D Biol Crystallogr, (2002) 58(Pt 11): p. 1955-7) of known crystal stractures, representing dimeric and teframeric quaternary folds of known CD As.
  • the CEMl 5 sequence was modeled manually onto three dimensional template using the computer graphics package O (Jones et al.
  • trancations are expressed.
  • truncation products of the form CD1-PCD1 or CD2-PCD2 would preclude folding of structures depicted in 10a, 10b and lOd, whereas model 10c could fold, leaving open the possibility that either CD1-PCD1 or CD2-PCD2 is sufficient to suppress viral infectivity. Therefore, anti- HEV-l therapeutics can be designed that disrupt Vif suppression of catalytic activity at either a single CD or both CD1 and CD2 simultaneously. The results of such mutations provide feedback, allowing a more rigorous refinement of the model by use of Modeller Attorney Docket Number 21108.0035P1
  • Vif is known to have binding affinity for both viral RNA genomes and a variety of viral and cellular proteins (Simon et al. (1996) J. Virol. 70 (8):5297-5305; Khan et al. (2001) J. Virol. 75(16):7252-7265; Henzler et al. (2001) J. Gen Virol. 82: p. 561-573). Vif also can forms homodimers and homoteframers through its proline rich domain (Yang et al. (2002) J. Biol Chem. 278(8):6596-6602).
  • Example 1 The infectivity assay in the context of Vif minus pseudotyped viruses and 293 T cells either lacking or expressing CEMl 5 is found in Example 1.
  • An assay was developed using VSV G-protein pseudotyped lentiviral particles that confirmed the inhibitory effect of CEMl 5 on the infectivity of vif+ and vif- HEV-l particles and is amenable to the rapid demarcation of the regions of HEV-l DNA (or RNA) that is the target for CEMl 5 catalytic activity.
  • Vif interacts with CEM15 and induces its poly-ubiquitination and degradation through the proteosome, thereby reducing the abundance of CEMl 5 and promoting viral infectivity. It has been discovered that Vif homodimers were required for Vif s interaction with CEMl 5 (Yang et al. J Biol Chem. 278(8): 6596-602 (2003) and US Patent 6,653,443, herein incorporated by reference in their entirety).
  • the CEM15 binding site of Vif can be similarly targeted, thereby achieving the same goal.
  • Peptides or small molecules that bind the CEM15 binding site of Vif can similarly suppress Vif s effect on CEMl 5.
  • the Vif antagonists and methods for screening the same can be agents that block the CEMl 5 linker or the CEMl 5 binding site.
  • Vif antagonists include agents that block the Vif-mediated polyubiquitination of CEM15. Vif interaction with CEM15 mediates CEM15's interaction with the polyubiquitination machinery, thereby leading to CEMl 5 Attorney Docket Number 21108.0035P1
  • polypeptides comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • polypeptide binds Vif and blocks ubiquitination of CEM15. Also disclosed is a polypeptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • polypeptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more or more contiguous amino acid residues of a CEM15 binding domain on Vif, wherein the polypeptide blocks CEMl 5-Vif interaction, as well as a method of blocking CEMl 5- Vif interaction comprising contacting Vif or CEM15
  • CEMl 5 contains a Gag binding domain. This binding domain allows for the CEMl 5 to be packaged into the virus. Vif, however, can block packaging from occurring. Thus, peptide mimetics resemblying the protein sequence of CEMl 5 that binds to Gag and the the CEMl 5 protein sequence that binds to Vif can interact with
  • a polypeptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous amino acid residues of a Gag protein,
  • polypeptide binds CEMl 5 and promotes CEMl 5 binding to viral RNA. Also disclosed is a method of promoting CEMl 5 binding to viral RNA comprising contacting CEMl 5 with the polypeptide disclosed herein.
  • CEMl 5 significantly reduced the level of pseudotyped HEV-l particles lacking Vif.
  • the reduced viral particle production is the result of a selective suppression of viral RNA leading to reduction in essential HEV-l 20 proteins. These effects were not observed when Vif was expressed due to the marked reduction of CEMl 5.
  • CEMl 5 was required to deplete viral particle production its deaminase function was not necessary. The data indicate an antiviral mechanism in producer cells which is potentially significant late during the viral life cycle that involves directly or indirectly the RNA binding ability of CEMl 5 and 25 does not require virion incorporation of CEMl 5 deaminase activity during viral replication.
  • agents that enhance CEMl 5 selective binding to viral RNA, leading to viral RNA distraction result in a reduction in viral particle production and a reduced viral burden for the subject.
  • Peptides conesponding to the portion of Gag protein sequence that binds to CEMl 5 can provide specificity to CEMl 5 for viral 30 RNA binding by CEMl 5.
  • TAT transduction of these peptide mimetics activates CEMl 5 antiviral activity within cells.
  • a pharmaceutical carrier includes mixtures of two or more such caniers, and the like.
  • Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value.
  • basal levels are normal in vivo levels prior to, or in the absence of, addition of an agent such as a Vif antagonist or another molecule or ligand.
  • test compound is defined as any compound to be tested for its ability to bind to a Vif molecule, a deoxycytidine deaminase molecule, or a cytidine deaminase molecule.
  • test compounds include, but are not limited to, small molecules such as K + , Ca 2+ , Mg 2+ Fe 2+ or Fe 3+ , as well as the anions SO 4 2" , H 2 PO " (or H 3 PO 4 ) and NO " .
  • test compounds include drugs, molecules, and compounds that come from combinatorial libraries where thousands of such ligands are screened by drag class.
  • subject is meant an individual.
  • the subject is a mammal such as a primate, and, more preferably, a human.
  • the term “subject” can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).
  • control levels or "control cells” are defined as the standard by which a change is measured, for example, the confrols are not subjected to the experiment, but are instead subjected to a defined set of parameters, or the controls are based on pre- or post-treatment levels.
  • contacting is meant an instance of exposure of at least one substance to another substance.
  • contacting can include contacting a substance, such as a cell, or cell to a test compound described herein.
  • a cell can be contacted with the test compound, for example, by adding the protein or small molecule to the culture medimn (by continuous infusion, by bolus delivery, or by changing the medium to a medium that contains the agent) or by adding the agent to the extracellular fluid in vivo (by local ⁇
  • a cell or group of cells is determined by the time the test compound is present at physiologically effective levels or at presumed physiologically effective levels in the medium or extracellular fluid bathing the cell.
  • a virally infected cell e.g., a, HEV infected cell
  • a cell at risk for viral infection e.g., before, at about the same time, or shortly after HEV infection of the cell
  • Treatment or “treating” means to administer a composition to a subject or a system with an undesired condition or at risk for the condition.
  • the condition can be
  • any pathogenic disease, autoimmune disease, cancer or inflammatory condition any pathogenic disease, autoimmune disease, cancer or inflammatory condition.
  • the effect of the administration of the composition to the subject can have the effect of but is not limited to reducing the symptoms of the condition, a reduction in the severity of the condition, or the complete ablation of the condition.
  • an effective amount is meant a therapeutic amount needed to achieve the 15 desired result or results, e.g., editing nucleic acids, interrupting CEMl 5-vif binding, reducing viral infectivity, inducing class switch recombination, somatic hypermutation, enhancing or blunting physiological functions, altering the qualitative or quantitative nature of the proteins expressed by cell or tissues, and eliminating or reducing disease causing molecules and/or the mRNA or DNA that encodes them, etc.
  • inhibition means to reduce activity as compared to a control (e.g., activity in the absence of such inhibition). It is understood that inhibition or suppression can mean a slight reduction in activity to the complete ablation of all activity.
  • An “inhibitor” or “suppressor” can be anything that reduces the targeted activity. For example, suppression of CEMl 5-Vif binding by a disclosed
  • composition can be determined by assaying the amount of CEMl 5-Vif binding in the presence of the composition to the amount of CEMl 5-Vif binding in the absence of the composition and by decrease and increase (respectively) in viral infectivity. In this example, if the amount of CEMl 5- Vif binding is reduced in the presence of the composition as compared to the amount of CEMl 5-Vif binding in the absence of the
  • composition can be said to suppress the CEMl 5-Vif binding.
  • systems can be, for example, cells, columns, or batch processing containers (e.g., Attorney Docket Number 21108.0035P1
  • a system is a set of components, any set of components that allows for the steps of the method to performed.
  • a system will comprise one or more components, such as a protein(s) or reagent(s).
  • a protein(s) or reagent(s) such as a protein(s) or reagent(s).
  • One type of system disclosed would be a cell that comprises both Vif and a test compound, for example.
  • Another type of 5 system would be one that comprises a cell and an infective unit (e.g., an HEV unit).
  • a third type of system might be a chromatography column that has CEMl 5, AED, or other deaminase or putative deaminase, bound to the column.
  • virally infected mammalian cell system or “virally infected” is meant an in vitro or in vivo system infected by a virus.
  • a virus can include mammalian
  • HEV infectivity or “viral infectivity” is meant the capacity of an in vitro or in vivo system to become infected by an virus (e.g., an HEV viras).
  • Vif antagonist any molecule or composition that counteracts, reduces, suppresses, inhibits, blocks, or hinders the activity of a Vif molecule or a
  • Vif dimerization antagonists which reduce, suppress, inhibit, block, or hinder the dimerization of Vif. Any time a "Vif antagonist” is mentioned, this includes Vif dimerization antagonists. Also included are agents that block Vif binding to the CEMl 5, agents that block Vif-mediated polyubiquitination of CEMl 5, and the like.
  • cytidine deaminase activator any molecule or composition that enhances or increases the activity of a cytidine deaminase molecule or a fragment thereof.
  • cytidine deaminase activator is also meant deoxycytidine deaminase activator, ARP activator, or any related molecule.
  • deoxycytidine deaminase activator any molecule or
  • composition that enhances or increases the activity of a deoxycytidine deaminase molecule or a fragment thereof.
  • ARP activator is meant any molecule or composition that enhances or increases the activity of an APOBEC-1 Related Protein molecule or a fragment thereof.
  • a "cytidine deaminase-positive cell” means any cell that expresses one ore 30 more cytidine deaminases or deoxycytidine deaminases. Such express can be naturally occurring or the cell can include an exogenous nucleic acid that encodes one ore more selected deaminases.
  • the method of screening for Vif antagonists comprises contacting a Vif molecule with a test compound; detecting binding between the Vif molecule and the test compound or detecting other desired interactions (such as CEM15-Vif binding or binding of Vif with proteins of the polyubiquitin machinery or block Gag interaction with CEM15); and screening the test compound that binds the Vif molecule or display another interaction for suppression of viral infectivity. Suppression of viral infectivity by the test compound indicates the test compound is a Vif antagonist. For the identification of Vif antagonists, it is not necessary to know whether Vif interacts with CEMl 5 or other viral or cellular proteins nor is it necessary to know the region(s) of Vif that is required to inhibit CEMl 5 activity.
  • CEMl 5 molecule and the test compound or detecting other desired interactions such as CEMl 5-Vif binding or binding of Vif with proteins of the polyubiquitin machinery or block Gag interaction with CEMl 5; and screening the test compound that binds the CEMl 5 molecule for its ability to block binding of Vif with the CEMl 5 or to suppress viral activity.
  • An agent that blocks binding of Vif to CEM15 or displays other desired interactions is a Vif antagonist, which can be further tested for its ability to suppress viral infectivity.
  • suppression means to reduce activity as compared to a control (e.g., activity in the absence of such inhibition or suppression). It is understood that inhibition or suppression can mean a slight reduction in activity to the complete ablation of all activity.
  • An "inhibitor” or “suppressor” can be anything that reduces activity.
  • suppression of CEMl 5- Vif binding by a disclosed composition can be determined by assaying the amount of CEMl 5-Vif binding in the presence of the composition to the amount of CEMl 5-Vif binding in the absence of the composition. In this example, if the amount of CEMl 5-Vif binding is reduced in the presence of the composition as compared to the amount of CEMl 5- Vif binding in the Attorney Docket Number 21108.0035P1
  • the composition can be said to suppress the CEMl 5-Vif binding.
  • This assay can be extended to include Vif + proviral DNA confrols and the use of deaminase inactivated CEMl 5 mutants in stable 293T cell lines.
  • the assay is also amenable to the use of several existing HEV-l proviral isotyped vectors that are deleted for different regions and different amounts of the HEV-l genome, as well as to other retroviruses. Deleted genes can be provided in trans by co-transfection of suitable expression plasmids.
  • a comprehensive examination of viral proteins and host tRNA Lys3 derived from Vif- virions revealed no significant biochemical or priming defects (Gaddis et al. J. Virol 77(10):5810-5820, 2003.) Dissection of such modifications can be performed in pseudotype viral assays in which key infectivity factors can be rapidly identified and assayed.
  • the screening assay described herein is useful for detecting Vif antagonists, deoxycytidine deaminase activators, or cytidine deaminase activators. These can block, prevent, or inhibit dimerization of Vif, block the Vif binding site for CEMl 5 or change the charge of CEMl 5 or compete with the CEM15/Vif binding sites to block or inhibit binding, block polyubiquitination, enhance CEMl 5 binding to viral RNA, or block Gag interaction with CEMl 5.
  • each cytidine deaminase activator, deoxycytidine deaminase activator, ARP activator, and Vif antagonist test compound can be tested by treating one or more of the cell types expressing a cytidine deaminase or deoxycytidine deaminase, or ARP, with each test compound and by infecting them with HIV-1 pseudotyped viras (or another refrovirus, or HCV or HBV, for example) containing GFP as described above.
  • HIV-1 pseudotyped viras or another refrovirus, or HCV or HBV, for example
  • supernatants containing viral particles can be added to HeLa cells to test their infectivity, as evidenced by the appearance of green fluorescent cells in FACS analysis as described above. Reduction or elimination of green fluorescent cells relative to that observed in infections from producer cells that were not treated with cytidine deaminase activators or Vif antagonists are scored as a positive identification of cytidine deaminase activators, deoxycytidine deaminase activators, or Vif antagonist test compounds.
  • Vif antagonists, deoxycytidine deaminase activators, or cytidine deaminase activators enable the normal cellular amounts of CEMl 5 to mutate HEV-l, HCV, HB V, MLV, or any other retrovirus, to the extent that the virus cannot reproduce itself and therefore cannot elicit a productive infection.
  • Vif antagonists enable CEMl 5 to mutate viral sequence at the level of first sfrand DNA synthesis and the resultant dC to dU change is templated during second strand DNA synthesis as dG to dA changes. The frequency of these changes is significantly greater than the mutation rate of reverse transcriptase and consequently the mutations in the retro viral genome affect numerous coding sequences at numerous positions, thereby rendering the viras nonfunctional (incapable of producing infectious virions).
  • the screening methods disclosed herein can be used with a high throughput screening assay, for example.
  • the high throughput assay system can comprise an immobilized anay of test compounds.
  • the Vif molecule or the cytidine deaminase molecule can be immobilized.
  • There are multiple high throughput screening assay techniques that are well known in the art for example, but not limited to, those described in Abriola et al., J Biomol. Screen 4:121-127, 1999; Blevitt et al., J Biomol. Screen 4:87-91, 2000; Hariharan et al., J Biomol. Screen 4:187-192, 1999; Fox et al., J Biomol.
  • Vif molecule deoxycytidine deaminase activator or cytidine deaminase activator can be linked to a reporter, such as luciferase, GFP, RFP, or FITC, for example.
  • Glow luminescence assays have been readily adopted into high throughput screening facilities because of their intrinsically high sensitivities and long-lived signals.
  • the signals for chemiluminescence, bioluminescence, and colorimetric systems Attorney Docket Number 2110S.0035P1
  • luciferase and beta-galactosidase reporter genes or for alkaline phosphatase conjugates are often stable for several hours.
  • luminescence in the whole plate by imaging with a CCD camera, similar to the way that calcium responses are read by calcium-sensitive fluorescent dyes in the FLEPR or FLEPR-384 instruments.
  • Other luminescence or fluorescence imaging systems include LEADSEEKER from AMERSHAM, the WALLAC VIEWLUX TM ultraHTS microplate imager, and the MOLECULAR DEVICES CLIPR imager.
  • NORTHSTAR TM HTS Workstation This instrument is able to rapidly dispense liquid into 96-well or 384-well microtiter plates by an external 8 or 16-head dispenser and then can quickly transfer the plate to a CCD camera that images the whole plate.
  • the total time for dispensing liquid into a plate and transferring it into the reader is about 10
  • Vif molecule and the reporter can also form a chimera.
  • Purified recombinant Vif e.g., HA/6His or Vif-CMPK-HA 6His, where CMPK is chicken muscle pyruvate kinase
  • FITC fluorescein isothiocyanate
  • fusion protein of Vif and GFP see diagram below
  • the Vif molecule can be represented by SEQ ID NO: 7, and the HA domain of the molecule can be represented by SEQ ED NO: 46.
  • the Vif- HA/6-His molecule can be represented by SEQ ED NO: 54 as follows:
  • Vif-GST Designates a TEV protease cleavage site (or other appropriate protease cleavage site) where a proteolytic cleavage can be performed on recombinant Vif- CMPK so that Vif may be purified free of CMPK prior to its conjugation to FITC.
  • Vif with or without CMPK may be produced depending on which protein produces the highest yield of soluble protein.
  • a similar strategy can be used for Vif-GST, in which GST is glutathione-S-fransferase fused to the Vif N-terminus.
  • Vif can be freed from the GST affinity tag by cleavage with PreScissionTM protease, and is then suitable for fluorescein labeling.
  • Regions 6His and HA are not drawn to scale.
  • GFP can also be used in conjunction with the Vif molecule.
  • Vif-GFP would not require a protease cleavage site due to its fluorescence; hence GFP- Vif would not require FITC conjugation.
  • cytidine deaminase or deoxycytidine deaminase activator or ARP activator HTS screening Vif has been substituted with CEMl 5 in all of the constructs listed above. 97.
  • the Vif-TEV-CMPK-HA 6-His molecule can be represented by SEQ ID
  • Vif-TEV-EGFP-HA/6-His molecule can be represented by SEQ ED
  • deaminases, ARPs, or cytidine deaminases can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually, all of the compounds listed above.
  • any number of chemical extracts or compounds can be screened using the exemplary methods described herein.
  • extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis
  • Synthetic compound libraries are commercially available, e.g., from Brandon Associates (Menimack, NH) and Aldrich Chemical (Milwaukee, WI).
  • a method of screening for cytidine deaminase activators comprising: contacting a cytidine deaminase molecule with a test compound; detecting binding between the cytidine deaminase molecule and the test compound; and screening the test Attorney Docket Number 21108.0035P1
  • the cytidine deaminase molecule can be CEM15. Therefore, the cytidine 5 deaminase activator can be a CEMl 5 activator.
  • the selected CEMl 5 function can be an increase, decrease, or any modification in the activity of the CEMl 5 or modifications in CEMl 5 interaction with other proteins (such as Vif) that modulate CEMl 5 deaminase activity.
  • the activity of CEMl 5, such as deoxycytidine to deoxyuridine mutation in the first strand of cDNA can be increased upon binding of a test 10 compound, thereby decreasing or suppressing viral infectivity.
  • the activity of CEMl 5 can be decreased, wherein the test compound binds CEMl 5 and the cytidine to uridine editing of mRNA or deoxycytidine to deoxyuridine mutation of DNA is inhibited or suppressed.
  • a decrease in CEMl 5 activity can decrease its cancer promoting activity, or reduce cancer phenotype, in vitro or in vivo.
  • An example of a 15 decrease in cancer promoting activity in the presence of compomids that bind CEMl 5 is found in breast cancer.
  • test compound to suppress viral infectivity can be measured by contacting the test compound with a cytidine deaminase molecule in the presence of Vif and a virus.
  • assays disclosed herein are useful 20 for detecting Vif antagonists, deoxycytidine deaminase activators, or cytidine deaminase activators. These can block, prevent, or inhibit dimerization of Vif, block the Vif binding site for CEMl 5 or change the charge of CEMl 5 or compete with the CEMl 5/ Vif binding sites to block or inhibit binding, block polyubiquitination, enhance CEMl 5 binding to viral RNA, or block Gag interaction with CEMl 5. 25 102.
  • the CEMl 5 function can be, but is not limited to, its cytidine to uridine editing of RNA, or its deoxycytidine to deoxyuridine mutation of DNA, or its suppression of viral activity, or its activity on cancerous or precancerous cells.
  • An “increase in CEMl 5 activity” is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold increase in the function of the 30 CEM15.
  • a "decrease in CEM15 activity” is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold decrease in the function of the CEM15.
  • the cytidine deaminase molecule can also be APOBEC-1. Therefore, the cytidine deaminase activator is an APOBEC-1 activator.
  • the activity of APOBEC-1 can be increased such that the levels of apoB48 are increased due to cytidine to uridine editing of apoB mRNA and the levels of apoBlOO are consequently decreased as compared to a control level.
  • Increasing APOBEC-1 activity can reduce atherogenic risk by promoting the activity of TAT- APOBEC-1 or the activity of APOBEC-1 expression from a transgene.
  • the activity of APOBEC-1 can be decreased by binding of APOBEC-1 and the test compound, wherein the cytidine to uridine editing of mRNA or deoxycytidine to deoxyuridine mutation of DNA is inhibited or suppressed.
  • An example of the decrease in cancer promoting activity in the presence of compounds that bind CEMl 5 is found in colon or rectal cancers.
  • the APOBEC-1 function can be, but is not limited to, its cytidine to uridine editing of RNA, or its deoxycytidine to deoxyuridine mutation of DNA, or the increased levels of apoB48 or decreased levels of apoBlOO as compared to a control, or its activity on cancerous or precancerous cells.
  • An "increased levels of apoB48" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold increase in the level of apoB48 as compared to a confrol.
  • a “decreased level of apoBlOO” is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold decrease in level of apoBlOO as compared to a control.
  • the cytidine deaminase molecule can also be AED. Therefore, the cytidine deaminase activator is an AED activator.
  • the activity of AED can be increased such that the levels of cytidine to uridine editing or the levels of deoxycytidine to deoxyuridine mutation are increased and the subsequent and consequent class switch recombination and or somatic hypermutation within the immunoglobulin locus of genes within B lymphocytes is increased.
  • Increasing AID activity can enhance the immune response in individuals that are immunocompromised or have become immunodepressed.
  • a D activity for example, the AID activity that promotes class switch recombination
  • the activity of AED can be decreased such that the levels of cytidine to Attorney Docket Number 21108.0035P1
  • uridine RNA editing or deoxycytidine to deoxyuridine mutation are decreased (for example, the AED activity that promotes somatic hypermutation), thereby reducing cancer promoting activity or cancer phenotype.
  • An example of the decrease in cancer promoting activity in the presence of compounds that bind AJJD is found in the freatment of B cell lymphomas that express or overexpress AED, thereby creating inappropriate AED edited mRNAs or AED mutated DNA sequences, or mutant forms thereof.
  • These cells may or may not have undergone class switch recombination or somatic hypermutation.
  • the AED function can be, but is not limited to, its cytidine to uridine editing of RNA, or its deoxycytidine to deoxyuridine mutation of DNA, or the promotion of antibody diversity produced by lymphocytes as compared to antibody production by control lymphocytes, or its activity on cancerous or precancerous cells.
  • “Promotion of antibody diversity” is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold increase in diversity of antibodies as compared to confrol lymphocytes.
  • a “decreased level of AED” is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100- fold, or 1000-fold decrease in level of AED as compared to a control.
  • the cytidine deaminase molecule can also be another ARP listed in Table 1. Therefore, the cytidine deaminase activator is an ARP activator.
  • the activity of ARP can be increased such that the levels of cytidine to uridine editing or the levels of deoxycytidine to deoxyuridine mutation are increased and the subsequent encoded macromolecule affected by RNA editing or DNA mutation and the physiological process dependent on that native sequence of the affected macromolecule is modulated.
  • RNA editing and DNA mutations induced by ARPs can have health promoting activities when appropriate regulated or disease causing activities when dysregulated.
  • the ARP function can be, but is not limited to, the cytidine to uridine editing of RNA, or the deoxycytidine to deoxyuridine mutation of DNA, or the promotion of health-promoting or disease-causing pathways.
  • the cytidine deaminase can also be linked to a reporter, such as luciferase, GFP, RFP, or FITC, for example.
  • the cytidine deaminase or Vif and the reporter can also form a chimera, as disclosed above.
  • the cytidine deaminase molecule can be CEMl 5, AED, APOBEC-1, or any other ARP molecule.
  • the sequences conesponding to CEM15, AID, and APOBEC-1 are SEQ ED NOS: 1, 3, and 5, respectively.
  • the conesponding nucleic acid sequences are SEQ ED NOS: 2, 4, and 6, respectively.
  • compositions e.g., Vif, cytidine deaminase, or their variants or fragments thereof
  • Vif, cytidine deaminase, or their variants or fragments thereof can be used as discussed herein as either reagents in micro anays or as reagents to probe or analyze existing microanays.
  • the compositions can also be used in any known method of screening assays, related to chip/micro anays.
  • the compositions can also be used in any known way of using the computer readable embodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions.
  • the effectiveness of the Vif antagonists or the cytidine deaminase activator can be assessed by detecting deaminase activity.
  • levels of edited viral RNA and/or mutated (edited) viral DNA wherein elevated levels of edited viral RNA or mutated (edited) viral DNA indicate enhanced deaminase activity.
  • levels of cellular RNA and DNA deaminases comprising by detecting levels of edited cellular RNA and/or mutated (edited) cellular DNA.
  • An isolating step can comprise incubating the mixture with molecule comprising Vif or a fragment or derivative thereof. 0
  • 114 Disclosed are methods of identifying an inhibitor or suppressor of an interaction between a deaminase and a viral infectivity factor (e.g., CEMl 5 and Vif, respectively) comprising incubating a library of molecules with the viral infectivity factor to form a mixture, and identifying the molecules that disrupt the interaction between the deaminase and the viral infectivity factor.
  • the interaction disrupted can comprise an interaction between the viral infectivity factor and an amino acid of deaminase.
  • An isolation step can comprise incubating the mixture with a molecule comprising a cytidine deaminase or fragment or derivative thereof.
  • Compositions 115 Disclosed are Vif antagonists identified by the screening methods.
  • cytidine deaminase activators identified by the screening methods. Also disclosed are deoxycytidine deaminase activators identified by the screening methods. Also disclosed are ARP activators identified by the screening methods.
  • the agents can function by interacting with Vif (e.g., Vif antagonist) or interacting with deoxycytidine deaminase or cytidine deaminase (e.g., cytidine deaminase activator).
  • Vif e.g., Vif antagonist
  • the Vif antagonist can bind or otherwise interact indirectly with Vif, thereby inhibiting its interaction with CEMl 5.
  • the cytidine deaminase activator or deoxycytidine deaminase activator can bind, or otherwise interact, with a cytidine deaminase or deoxycytidine deaminase, thereby enhancing the normal activity of the cytidine deaminase or deoxycytidine deaminase.
  • a cytidine deaminase activator can interact with CEMl 5 and enhance the binding of CEMl 5 to a virus.
  • a cytidine deaminase activator can interact with the binding of Vif to a CEMl 5 molecule, thereby suppressing the activity of Vif, and indirectly enhancing CEMl 5 binding to HEV.
  • the Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators of the invention can be modified to enhance suppression of viral activity or to lower biotoxicity. Such modification can 05/023
  • Vif antagonist or cytidine deaminase molecule can be modified following Lipinski's Rule of Five.
  • P the concentration of the compound in water divided by the concentration of the compound in 1 octanol
  • compositions of the invention are chimeric proteins.
  • chimeric protein is meant any single polypeptide unit that comprises two distinct polypeptide domains joined by a peptide bond, optionally by means of an amino acid linker, or a non-peptide bond, wherein the two domains are not naturally occurring within the same polypeptide unit.
  • chimeric proteins are made by expression of a cDNA construct but could be made by protein synthesis methods known in the art. These chimeric proteins are useful in screening compounds, as well as with the compounds identified by the methods disclosed herein.
  • the compositions disclosed herein can also be fragments or derivatives of a naturally occuning deaminase or viral infectivity factor.
  • a “fragment” is a polypeptide that is less than the full length of a particular protein or functional domain.
  • derivative or “variant” is meant a polypeptide having a particular sequence that differs at one or more positions from a reference sequence.
  • the fragments or derivatives of a full length protein preferably retain at least one function of the full length protein.
  • a fragment or derivative of a deaminase includes a fragment of a deaminase or a derivative deaminase (e.g., APOBEC-1, AED, CEMl 5, or Attorney Docket Number 21108.0035P1
  • the fragment or derivative can include a Zinc-Dependent Cytidine Deaminase domain or can include 20, 30, 40, 50, 60, 70 80, 90% similarity with the full length deaminase.
  • the fragment or derivative can include conservative or non-conservative amino acid substitutions.
  • the fragment or derivative can include a linker sequence joining a catalytic domain (CD) to a pseudo-catalytic domain (PCD) and can have the domain stracture CD-PCD-CD-PCD or any repeats thereof.
  • the fragment or derivative can comprise a CD.
  • fragments or derivatives are identified by structure-based sequence alignment (SBSA) as shown herein. See Figure 6b that reveals the consensus stractural domain attributes of APOBEC-1 and ARPs ( Figure 6c).
  • the fragment or derivative optionally can form a homodimer or a homotetramer.
  • chimeric proteins wherein the deaminase domain is a fragment or derivative of CEMl 5 having deaminase function.
  • Deaminases include deoxycytidine deaminase, cytidine deaminase, adenosine deaminase, RNA deaminase, DNA deaminase, and other deaminases.
  • the deaminase is APOBEC-1 (see international patent application designated PCT/US02/05824, which is incorporated herein by reference in its entirety for APOBEC-1, chimeric proteins related thereto, and uses thereof) (Gen Bank Accession # NPJ301635), REE (see U.S. Pat. No. 5,747,319, which is incorporated herein by reference in its entirety for REE and uses thereof), or REE-2 (see U.S. Pat. No. 5,804,185, which is incorporated herein by reference in its entirety for REE-2 and uses thereof).
  • Deaminases as described herein can include the following structural features: three or more CDD-1 repeats, two or more functional CDD-1 repeats, one or more zinc binding domains (ZBDs), binding site(s) for mooring sequences, or binding sites for auxiliary RNA binding proteins.
  • Deaminases optionally edit viral RNA, host cell mRNA, viral DNA, host cell DNA or any combination thereof.
  • One deaminase described herein is CEMl 5.
  • CEMl 5 is homologous to Phorbolin or APOBEC-3G (see, for example, Accession #NP_068594). The names CEMl 5 and APOBEC-3G can be used interchangeably.
  • CEMl 5 reduces retro viral infectivity as an RNA or DNA editing enzyme.
  • deaminating function is meant a deamination of a nucleotide (e.g., cytidine, deoxycytidine, adenosine, or deoxyadenosine). Deaminating function is
  • the Vif fragment or derivative thereof has at least 20, 30, 40, 5 50, 60, 70, 80, or 90 % amino acid similarity with the Vif molecule of SEQ ID NO: 7.
  • the APOBEC-1 fragment or derivative thereof has at least 20, 30, 40, 50, 60, 70, 80, or 90 % amino acid similarity with the APOBEC-1 molecule of SEQ ED NO: 5.
  • the AID fragment or derivative thereof has at least 20, 30, 40, 50, 60, 70, 80, or 90 % amino acid similarity with the AED molecule of SEQ ID NO: 3.
  • the CEM15 fragment or derivative has at least 20, 30, 40, 50, 60, 70, 80, or 90 % amino acid similarity with the CEMl 5 molecule of SEQ ED NO: 1.
  • SEQ ED NO: 2 sets forth a particular nucleic acid sequence that encodes a CEMl 5
  • SEQ ED NO: 1 sets forth particular sequences of the proteins encoded by those nucleic acids.
  • SEQ ED NO: 2 sets forth a particular nucleic acid sequence that encodes a CEMl 5
  • SEQ ED NO: 1 sets forth particular sequences of the proteins encoded by those nucleic acids.
  • 25 NOS: 4, 6, and 8 sets forth particular nucleic acid sequences that encode an ALD, an APOBEC-1, and a Vif protein, respectively, and SEQ ED NOS: 3, 5, and 7 sets forth particular sequence of the proteins encoded by those nucleic acids.
  • variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
  • nucleic acids such as genes.
  • the similarity can be calculated after aligning the two sequences so that the similarity is at its highest level.
  • Another way of calculating similarity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the alignment algorithm of Needleman and Wunsch, J. Mol Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WE), or by inspection.
  • a sequence recited as having a particular percent similarity to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above.
  • a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent similarity to the second sequence as calculated by any of the other calculation methods.
  • a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent similarity to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods.
  • a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated similarity percentages). 5 129.
  • Other structural similarities aside from sequence similarity are also disclosed. For example, homology, as noted by similar secondary and tertiary stracture, can be analyzed as taught herein. Homologous proteins may have minimal sequence similarity but have a homologous catalytic domain. Thus, deaminases as used herein maybe structurally similar based on the structure of the catalytic domain or other
  • Vif antagonists as well as cytidine deaminase activators, deoxycytidine deaminase activators, and ARP activators can be identified using variants and derivatives of cytidine deaminases, deoxycytidine deaminases, or Vif. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for
  • Immunogenic fusion protein derivatives such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein
  • variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a
  • substitutions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
  • substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
  • the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary RNA stracture.
  • substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are refened to as conservative substitutions.
  • Trp Tyr Tyr; Trp; Phe Val; He; Leu
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 3, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the stracture of
  • substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic
  • Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also may be desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the conesponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of ' the C-terminal carboxyl.
  • compositions disclosed herein can be used as targets in combinatorial chemistry protocols or other screening protocols to isolate molecules that possess desired functional properties related to inhibition of the CEMl 5-Vif, activation of cytidine deaminase or deoxycytidine deaminase, or antagonism of Vif activity. 137.
  • molecules that function like the disclosed molecules can be identified and used as discussed herein. For example, the knowledge that CEMl 5 interacts with Vif indicates targets for identifying molecules that will affect retroviral infectivity.
  • compositions and methods of making these compositions that bind (or interact with) cytidine deaminase molecules, such as CEMl 5.
  • the molecules enhance or suppress a cytidine deaminase or deoxycytidine deaminase function.
  • this knowledge can be used along with, for example, combinatorial chemistry techniques, identify molecules that function as desired, by for example, inhibiting or suppressing CEMl 5 and Vif binding, or mimic other cytidine deaminases or deoxycytidine deaminases.
  • Attorney Docket Number 21108.0035P1 Attorney Docket Number 21108.0035P1
  • compositions such as cytidine deaminases or deoxycytidine deaminases (e.g., CEM15, APOBEC-1, AED, and other ARPs) or Vif can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired
  • nucleic acids, peptides, and related molecules disclosed herein can be used as targets for the combinatorial approaches.
  • the molecules identified and isolated when using the disclosed compositions are also disclosed.
  • the products produced using the combinatorial or screening approaches that involve the disclosed compositions such as, CEMl 5, AED, APOBEC-1, ARPs, or Vif are also disclosed.
  • Such molecules include
  • Combinatorial chemistry includes but is not limited to all methods for isolating small molecules or macromolecules that are capable of binding either a small molecule or another macromolecule like Vif or cytidine deaminase (e.g., CEMl 5), typically in an iterative process.
  • a small molecule or another macromolecule like Vif or cytidine deaminase (e.g., CEMl 5), typically in an iterative process.
  • CEMl 5 cytidine deaminase
  • oligonucleotide molecules with a given function can be isolated from a complex mixture of random oligonucleotides in what has been refened to as "in vitro genetics" (Szostak, TLBS 19:89, 1992).
  • In vitro genetics Szostak, TLBS 19:89, 1992.
  • Combinatorial techniques are particularly suited for defining binding interactions between molecules and for isolating molecules that have a specific binding activity, often called aptamers when the 5 macromolecules are nucleic acids.
  • RNA molecule is generated in which a puromycin molecule is covalently attached to the 3 '-end of the RNA molecule.
  • An in vitro translation of this modified RNA molecule causes the conect protein, encoded by the RNA to be translated.
  • a peptdyl acceptor which cannot be extended, the growing RNA molecule
  • RNA is transcribed with puromycin at the 3 '-end, new peptide is translated and another functional round of selection is perfonned.
  • protein selection can be performed in an iterative manner just like nucleic acid selection techniques.
  • the peptide which is translated is controlled by the sequence of the RNA attached to the puromycin.
  • sequence can be anything from a random sequence engineered for optimum translation (i.e. no stop codons etc.) or it can be a degenerate sequence of a known RNA molecule to look for improved or altered function of a known peptide.
  • a peptide of choice for example a portion of Vif is attached to a DNA binding domain of a franscriptional activation protein, such as Gal 4.
  • a franscriptional activation protein such as Gal 4.
  • Combinatorial libraries can be made from a wide anay of molecules using a number of different synthetic techniques. For example, libraries containing fused 2,4-pyrimidinediones (United States patent 6,025,371) dihydrobenzopyrans
  • combinatorial methods and libraries include traditional screening methods and libraries as well as methods and libraries used in interative processes.
  • compositions including the Vif antagonists, deoxycytidine deaminase activators, ARP activators, and the cytidine deaminase
  • compositions comprising: The compounds disclosed herein can be used as targets in any molecular modeling program or approach.
  • the three-dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new compound will link to the target
  • CHARMm performs the energy minimization and molecular dynamics fimctions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular stracture. QUANTA allows
  • a compound that is identified or designed as a result of any of the disclosed methods can be obtained (or synthesized) and tested for its biological activity, e.g., competitive inhibition or suppression of CEMl 5-Vif binding or inhibition or suppression of retro viral infectivity.
  • compositions 156.
  • Disclosed are methods of interrupting viral infectivity comprising contacting an infected cell or a cell prior to infection with a Vif antagonist, under conditions that allow delivery of the antagonist into the cell, wherein the antagonist binds with a viral infectivity factor (Vif) or CEMl 5 to interrupt viral infectivity.
  • h terraption of viral infectivity may occur at different levels, including, for example, at the level of RNA on the incoming virus, on first or second strand cDNA, after dsDNA integration and/or on transcripts from the viral integrin.
  • interrupting viral infectivity is meant stopping or reducing the production of infective viral genomes.
  • HIV infectivity for example, is known to depend on a variety of proteins leading to the synthesis of double stranded DNA from single sfranded HEV RNA genome and the integration of HEV DNA into the host cell's chromosomal DNA from where it is expressed to form viral genomes and viral proteins necessary for virion production.
  • a Vif antagonist reduces the ability of virion Vif to inactivate cellular processes, thus allowing CEMl 5 to effectively mutate HEV or alters its replication and chromosomal integration by affecting the editing of a cellular mRNA encoding a protein that blocks the production of infectious HEV.
  • Vif antagonists, deoxycytidine deaminase activators, and cytidine deaminase activators described herein can work in a multitude of ways to interrupt viral infectivity. For example, they can block, prevent, or inhibit dimerization of Vif; block the Vif binding site for CEMl 5 or change the charge of CEMl 5 or compete with the CEM15/Vif binding sites to block or inhibit binding; block polyubiquitination; enhance CEMl 5 binding to viral RNA; or or block Gag interaction with CEMl 5.
  • compositions can be delivered to the target cells in a variety of ways.
  • the compositions can be delivered through elecfroporation, or through lipofection, or through calcium phosphate precipitation.
  • the delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
  • compositions can comprise, for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.
  • liposomes can further comprise proteins to facilitate targeting a particular cell, if desired.
  • Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract.
  • liposomes see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad.
  • the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.
  • a subject with a viral infection e.g., HEV infection
  • a viral infection e.g., HEV infection
  • administering comprising administering to the subject an effective amount of the Vif antagonist.
  • an agent described herein can be combined with various others therapies.
  • a subject with HEV may be treated concomitantly with protease inhibitors and other agents.
  • the compound can be in water soluble form, and can be administered by the various routes described throughout.
  • One example of administration is oral administration.
  • a cytidine deaminase activator is an agent that enhances the efficiency of editing. Additional genetic, pharmacologic, or metabolic agents or conditions also modulate the RNA or DNA editing or mutating function of the deaminase. Some of the conditions that modulate editing activity include: (i) changes in the diet, (ii) hormonal changes (e.g., levels of insulin or thyroid hormone), (iii) osmolarity (e.g., hyper or hypo osmolarity), (iv) ethanol, (v) inhibitors of RNA or protein synthesis and (vi) conditions Attorney Docket Number 21108.0035P1
  • the methods of the invention can comprise administering a cytidine activator to the subject and using other conditions that enhance the efficiency of mRNA editing function.
  • the cancer can be selected from the group consisting of lymphomas (Hodgkins and non-Hodgkins), B cell lymphoma, T cell lymphoma, myeloid leukemia, leukemias, mycosis fungoides, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic
  • lymphomas Hodgkins and non-Hodgkins
  • B cell lymphoma T cell lymphoma
  • myeloid leukemia leukemias
  • mycosis fungoides carcinomas
  • carcinomas of solid tissues squamous cell carcinomas
  • adenocarcinomas sarcomas
  • gliomas blastomas
  • neuroblastomas plasmacytomas
  • histiocytomas melanomas
  • adenomas hypo
  • tumours myelomas, AEDS-related lymphomas or sarcomas, metastic cancers, bladder cancer, brain cancer, nervous system cancer, squamous cell carcinoma of head and neck, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary
  • cancer 15 cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, hematopoietic cancers, testicular cancer, colo-rectal cancers, prostatic cancer, or pancreatic cancer.
  • condition to be treated is an infectious disease (e.g., a viral disease). Also disclosed are methods, wherein the viral
  • 20 infection can be selected from the list of virases consisting of Herpes simplex viras type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein-Ban viras, Varicella- zoster virus, Human herpesvirus 6, Human herpesvirus 7, Human herpesviras 8, Variola viras, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C viras, Hepatitis D viras, Hepatitis E viras, Rhinovirus, Coronavirus, Influenza virus
  • virases consisting of Herpes simplex viras type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein-Ban viras, Varicella- zoster virus, Human herpesvirus 6, Human herpesvirus 7, Human herpesviras 8, Variola viras, Vesicular stomatitis virus, Hepati
  • Influenza virus B Measles viras, Polyomaviras, Human Papilomaviras, Respiratory syncytial viras, Adenovirus, Coxsackie virus, Dengue viras, Mumps viras, Polioviras, Rabies viras, Rous sarcoma viras, Yellow fever viras, Ebola virus, Marburg viras, Lassa fever viras, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever
  • Rotavirus A Rotaviras B
  • Rotavirus C Sindbis virus
  • Simian Immunodeficiency ciras Human T-cell Leukemia viras type-1, Hantavirus, Rubella virus, Simian wo 2005/023 '
  • Immunodeficiency virus Human Immunodeficiency virus type-1, Vaccinia virus, SARS virus, and Human Immunodeficiency viras type-2.
  • the bacterial infection can include M. tuberculosis, M. bovis, M. bovis strain BCG, BCG
  • M. avium 5 subsfrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species, Yersinia pestis, Pasteur ella haemolytica, Pasteur ella multocida, other Pasteur ella species, Actinobacillus pleuropneumoniae,
  • Neiserria meningitidis Neiserria gonorrhea
  • Pseudomonas aeruginosa other Pseudomonas species
  • Haemophilus influenzae Haemophilus ducreyi
  • Clostridium tetani other Clostridium species
  • Yersinia enterolitica and other Yersinia species.
  • the 20 parasitic infection can include Toxoplasma gondii, Plasmodiumfalciparum,
  • Plasmodium vivax Plasmodium malariae, other Plasmodium species.
  • Trypanosoma brucei Trypanosoma cruzi
  • Leishmania major other Leishmania species.
  • Schistosoma mansoni other Schistosoma species.
  • Entamoeba histolytica Plasmodium vivax, Plasmodium malariae, other Plasmodium species.
  • Trypanosoma brucei Trypanosoma cruzi
  • Leishmania major other Leishmania species.
  • Schistosoma mansoni other Schistosoma species.
  • Entamoeba histolytica Entamoeba histolytica.
  • the fungal 25 infection can include Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicillium marnefft, and Alternaria alternata.
  • Vif antagonists, deoxycytidine deaminase activators, ARP activators, 30 and cytidine deaminase activators are of benefit to individuals who are infected as well as to those who have recently been infected or anticipate an exposure to the virus. As new virases are produced in individuals who are HEV positive, or positive 2005/023985 " .
  • Vif antagonist, deoxycytidine deaminase activator, ARP activator, or cytidine deaminase activator freatment will induce mutations as virus infects new cells. Many of the mutated viruses are destroyed by host cell DNA repair mechanism. Those mutated viras that integrate into chromosomal DNA are not able 5 to produce infectious viral particles. The overall effect is reduced viral shedding into body fluids and consequently a reduction in the probability that new contacts with infected individuals will be infectious. Therefore Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators reduce the production of infectious viruses in affected individuals thereby controlling the disease
  • Vif antagonists deoxycytidine deaminase activators, ARP activators, or cytidine deaminase activators can prevent a
  • the virus can be a retrovirus (e.g., HIV).
  • the virus can be an RNA virus.
  • the RNA virus can be selected from the
  • virases consisting of Vesicular stomatitis virus, Hepatitis A virus, Hepatitis C viras, Rhinoviras, Coronavirus, Influenza virus A, Influenza virus B, Measles viras, Respiratory syncytial viras, Adenovirus, Coxsackie viras, Dengue viras, Mumps viras, Polioviras, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus,
  • the ability to suppress viral infectivity can be measured by contacting the test compound with one or more cytidine deaminase-positive cells, in the presence
  • Cytidine deaminase positive cells are cells that express a cytidine deaminase molecule or fragment thereof, such as CEMl 5, APOBEC-1, AID, or ARPs. Attorney Docket Number 21108.0035P1
  • compositions can also be used diagnostic tools related to diseases that are susceptible to RNA or DNA editing, such as HEV, HCV, HBV, or MLV.
  • compositions can also be administered in vivo in 5 a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material maybe administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the 10 carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, fransdermally,
  • topical infranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. The latter
  • compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age,
  • compositions if used, are generally characterized by injection.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid Attorney Docket Number 21108.0035P1
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. 177.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin- coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in wliich the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concenfration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers 178.
  • Vif antagonists Delivery of the Vif antagonist, deoxycytidine deaminase activator, ARP activator, or cytidine deaminase activator compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Pharmaceutical carriers are Attorney Docket Number 21108.0035P 1
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. 180.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic freatment is desired, and on the area to be treated. Administration may be topically (including opthamalically, vaginally, rectally, infranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed compounds can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous earners include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral adnrinistration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions may potentially be administered as a
  • compositions are those
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual
  • Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Vif antagonists deoxycytidine deaminase activators, ARP activators, or cytidine deaminase activators that do not have a specific pharmaceutical function, but which may be used for tracking changes within cellular chromosomes or for the
  • 25 delivery of diagnostic tools for example can be delivered in ways similar to those described for the pharmaceutical products.
  • molecules such as Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators can be administered together with other forms of therapy.
  • the molecules such as Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators can be administered together with other forms of therapy.
  • the molecules such as Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators can be administered together with other forms of therapy.
  • the molecules such as Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators can be administered together with other forms of therapy.
  • the molecules such as Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine
  • TAT-fusion peptides are especially useful with the methods described herein, as they are rapidly internalized by lipid raft-dependent macropinocytosis and then able to escape.
  • dTAT-HA2 is also Attorney Docket Number 21108.0035P1
  • compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
  • Vif antagonists comprising identifying a Vif antagonist by the screening methods disclosed herein; and modifying the Vif antagonist to enhance suppression of viral infectivity. Methods of modifying the Vif antagonist are disclosed herein.
  • the Vif antagonist can be modified by a number of means, as disclosed above, such as using Lipinski's Rule of Five. Such modifications can include amino acid modifications, thereby producing variants and derivatives that enhance suppression of viral activity.
  • Vif antagonists and cytidine deaminase activators made by the methods described herein. 190.
  • Disclosed are methods of making a cytidine deaminase activator comprising identifying the cytidine deaminase activator; and modifying the cytidine deaminase activator to enhance the selected deaminase function of the modified cytidine deaminase activator as compared to the function of the unmodified cytidine deaminase activator.
  • Methods of modifying the cytidine deaminase activator are disclosed herein, such as using Lipinski's Rule of Five.
  • the cytidine deaminase activator can be modified by a number of means, as disclosed above.
  • Such modifications can include amino acid modifications, thereby producing variants and derivatives that enhance suppression of viral activity.
  • the same method can be used to make deoxycytidine deaminase activators and ARP activators. 191. "Suppression of viral activity" is defined as a 10%, 20%, 30%, 40%,
  • Viral activity includes, but is not limited to, viral reproduction, viral shedding, or viral infectivity.
  • Vif antagonist Also disclosed are methods of making a Vif antagonist, comprising identifying the Vif antagonist by the screening methods disclosed herein; and modifying the Vif antagonist to lower biotoxicity of the test compound. .
  • a method of making a cytidine deaminase activator comprising identifying the cytidine deaminase activator; and modifying the cytidine deaminase activator to lower biotoxicity of the modified cytidine deaminase activator as compared to the biotoxicity of the unmodified cytidine deaminase activator.
  • “Lower biotoxicity” is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold lowering of the biotoxicity of the test compound. Biotoxicity is defined as the toxicity of the compound to a cell or to a system, in vitro or in vivo.
  • Disclosed are methods of treating a subject comprising administering to the subject an inhibitor of viral infectivity (e.g., HEV infectivity), wherein the inhibitor reduces the interaction between a deaminase (e.g., CEMl 5) and a viral infectivity factor (Vif), and wherein the subject is in need of such freatment.
  • an inhibitor of viral infectivity e.g., HEV infectivity
  • the inhibitor reduces the interaction between a deaminase (e.g., CEMl 5) and a viral infectivity factor (Vif)
  • Vif viral infectivity factor
  • a composition for inhibiting the 15 interaction between a deaminase e.g., CEMl 5
  • a viral infectivity factor Vif
  • methods of manufacturing a composition for enhancing the activity of a deaminase such as CEM15, APOBEC-1, AED, or other ARPs.
  • Disclosed are methods of making a composition capable of inhibiting infectivity comprising admixing a compound with a pharmaceutically acceptable carrier, wherein the compound is identified by the methods described herein.
  • chips comprising nucleic acids that encode Vif, cytidine deaminases, deoxycytidine deaminases, ARPs, or fragments or variants thereof or where at least one address is such a nucleic acid. Also disclosed are chips where at least one address is an amino acid sequence for Vif, deoxycytidine deaminases, ARPs,
  • compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some enors and deviations should be accounted for. Unless indicated otherwise, parts are 10 parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.
  • U.S. Provisional Application No. 60/401,293 and PCT/US02/05824 are incorporated herein by reference in their entireties for the examples, methods, and compounds therein.
  • Human CEM15 (NP-068594; also known as MDS019, AAH24268) was amplified from total cellular RNA of the NALM-6 cell line (human B cell precursor leukemia) by RT-PCR.
  • Oligo-dT primed first-strand cDNA was amplified using Expand HiFi Taq DNA polymerase (Roche) with the following primers; '5 'A' CACTTTAGGGAGGGCTGTCC (SEQ ID NO: 10) and '3 'A* CTGTGATCAGCTGGAGATGG (SEQ ED NO: 11).
  • Thel366 bp product was reamplified with CEMl 5 specific PCR primers that included Nco ⁇ and IioI restriction
  • CTCCCATGGCAAAGCCTCACTTCAGAAACACAG SEQ ID NO: 12
  • '3'B' CTCCTCGAGGTTTTCCTGATTCTGGAGAATGGCCC SEQ ED NO: 13
  • the 1154bp PCR product was digested with EcoRL to remove potentially co-amplified highly homologous APOBEC3B/Phorbolin 3 (Q9UH17) sequences and the NcolVXhoI
  • HA haemagglutinin
  • the construction of the APOBEC-1 model is based upon the hypothesis that enzymes with a common, catalytic function (i.e. hydrolytic deamination of a nucleoside base) exhibit a common tliree-dimensional fold despite a low overall amino acid sequence identity (even at levels ⁇ 30%). This level of homology is often cited as the lower limit upon which one can reliably model the fold of a given polypeptide sequence (Burley, S.K. (2000) Nature Struct. Biol. 7:932-934.). However, the stractures of molecules with similar biological functions are known to be highly conserved even at low levels of primary stracture homology (Chothia et al. Embo J.
  • Cddl was amplified by PCR from Baker's yeast. The product was cloned into a pET-28a vector (Novagen) containing N-tenninal 6xHis using Ndel and EcoRI restriction sites; constructs were verified by DNA sequencing. BL21 CodonPlus (Stratagene) cells transformed with vector were grown at 37°C to an OD600 of 0.7 and induced with 1 mM EPTG at 30°C for 3 hours.
  • Bacterial pellets were resuspended in lysis buffer (50 mM Tris-Cl pH 8.0, 10 mM ⁇ -mercaptoethanol, 1 mg/ml lysozyme, lmM PMSF, 2 mM benzamidine and 5 ⁇ g/ml each of aprotinin, leupeptin and pepstatin A), lysed, and nuclease digested (0.5% Triton X-100, 2 mM ATP, 10 mM MgSO4, 33 ⁇ g/ml each of DNasel and RNasel) at 4°C.
  • lysis buffer 50 mM Tris-Cl pH 8.0, 10 mM ⁇ -mercaptoethanol, 1 mg/ml lysozyme, lmM PMSF, 2 mM benzamidine and 5 ⁇ g/ml each of aprotinin, leupeptin and pepstatin A
  • the 6xHis tagged protein was purified in batch with NiNTA agarose (Qiagen) utilizing the following wash, elution, and dialysis scheme: wash 7, 10 mM Tris-Cl pH 8.0, 100 mM KCl, 20 mM imidazole, 10% glycerol; wash 2, same as wash 1 including 1 M KCl; wash 3, repeat wash 1; elution, 10 mM Tris-Cl pH 8.0, 0.5 M KCl, 0.4 M imidazole, 10% glycerol; dialysis against 2x 2 liters 10 mM Tris-Cl pH 8.0, 120 mM NaCl, 1 mM DTT).
  • 6xHis tag Removal of the 6xHis tag was achieved by digestion for 16 hours at 20°C with 10 U biotinylated thrombin (Pierce). Protein was dialyzed against 20 mM HEPPS pH 8.0, 0.25 M KCl, 5% glycerol, and 4 mM DTT and concentrated to 6 mg/ml as estimated by Bradford assays (BioRad) using an Ulfrafree-4 spin cartridge (Millipore). Protein was utilized immediately for crystallization.
  • Crystals were grown at 20°C from well solutions of 16.5% (w/v) PEG monomethylether (MME) 5K, 450 mM NH 4 C1, 100 mM Na-succinate pH 5.5, 10 mM DTT and 1 mM NaN3 by use of the hanging drop vapor diffusion method. Four ⁇ l of well solution was added to an equal volume of protein. Crystals appeared in six days Attorney Docket Number 21108.0035P1
  • the positions of four zinc atoms were located by use of the program SOLVE v2.0, and phases were density modified by use of the program RESOLVE with 4-fold NCS averaging.
  • NCS averaged phases improved electron density maps significantly and allowed manual skeletonization by use of O. Additional NCS averaging with DM improved maps quality and allowed modeling of amino acids 4 to 136 in all four subunits. Upon addition of UMP, the C-terminal 6 amino acids are observed.
  • the model exhibits reasonable bond and angle deviations from ideal values (0.009A and 1.52o, respectively) as evaluated by PROCHECK. More than 89% of residues are in the allowed region of the Ramachandran Plot.
  • the edited nucleotide was modeled according to constraints derived from the known locations of CDA inhibitors in the template X-ray crystal stractures: 1 JKT (tetrahydrouridine ) and 1AF2 (3,4 dihydrouridine). Due to the known substrates of AID and APOBEC-1, DNA and RNA sequences were modeled as single-stranded. Additionally, the restraint that nucleotide bases flanking the edited/mutated sites maintain modest base stacking was imposed by adding additional distance restraints in the model calculation.
  • Cddl was PCR amplified using a 5' Cddl -specific primer and a 3' primer encoding the 19 amino acid E. coli "linker" extension and subcloned into the Ndel and EcoRI sites of pET28a (Novagen).
  • Glyl37 was converted to Ala using the QuikChange mutagenesis system (Stratagene) according to the manufacturer's protocols; other point mutations were constructed similarly.
  • PDB #1 AF2 was competent to edit under conditions similar to .
  • APOBEC-1 and Cddl in yeast were PCR amplified from genomic DNA and subcloned for yeast expression as described below.
  • a series of Cddl cliimeras were assembled by fusing together two Cddl polypeptide chains joined by a linker. The 5' monomers containing the appropriate C- terminal APOBEC-1 or E.
  • coli 19 amino acid linker were amplified and subcloned as described above.
  • the amino terminally foreshortened C-terminal monomer (missing helix ⁇ l based upon homology modeling) was PCR amplified using the wild type or Glu63 to Ala Cddl template and ligated as an EcoRlVXhoI fragment to the appropriate 5' monomer in pET28a.
  • the linking EcoRI site was mutagenized to restore the reading frame of the Cddl chimeras.
  • the ARP models suggest a re-structuring of the active site linker that makes the entire region spanning from 130 to 142 (human APOBEC-1 numbering) flexible in a manner that moves to accommodate large polymeric substrates such as RNA or DNA (See AID active site model bound to DNA 9-mer BELOW). Additional evidence of the importance of the linker sequence comes from mutagenesis on rat APOBEC-1 (highly homologous to human). When the 8 amino acid linker sequence of rat APOBEC-1 is replaced with the first 8 amino acids of the E. coli linker, the APOBEC-1 construct is unable to edit reporter apoB mRNA in the human hepatoma cell line HepG2 (Navaratnam, N. et al. (1998) JMB 275:695-714; Chester et al., 2003 EMBO J. 22, 3971-3982).
  • the hidden Markov modeling software SAM was trained with CDD 1 , APOBEC 1, APOBEC2, AID and phorbolin 1. This identified APOBEC3A, 3B, 3C, 3E, 3F, 3G, XP_092919, PHB1, XP_115170/XP_062365.
  • PHI-BLAST using the target pattern H[VA]-E-x-x-F-(x) 19-[17V]-[T/V]- [W/C]-x-x-S-W-[ST]-P-C-x-x-C (SEQ ID NO: 60) limited the search more and misses only the 3B (Phorbolin 2) variant AAD00089 in which a single codon change GAC/T (SEQ ID NO: 63) to GAA/G (SEQ ID NO: 64) changes the ZDD center HxE to HxA. This is either a sequencing enor or a significant SNP for psoriasis.
  • HPE....SPC C Also identifies a mouse gene homologous to hu APOBEC3G
  • IVS- ⁇ 3'5'apoB was created by ligation of the appropriate halves of the above molecules.
  • McArdle RH7777 cells were maintained as previously described (Sowden, M.P. et al., (1996) J. Biol. Chem. 271 :3011-3017.) and fransfected in six-well clusters with 2 ⁇ g of DNA using lipofectAM NE ® (Gibco BRL) according to the manufacturer's recommendations.
  • RNAs were harvested 48 h post-fransfection in TriReagent (Molecular Research Center, Cincinnati, OH, U.S.A.) and subjected to reverse-transcriptase (RT)-PCR for amplification of intron-containing or exonic apoB specific transcripts using appropriate PCR primers as previously described (Sowden, M., et al.
  • the poisoned-primer-extension assay relies on the annealing of a 32 P- end-labelled primer 3 ' of the editing site to the heat-denatured single-stranded PCR product. Extension of this primer using RT in the presence of dATP, dCTP, dTTP and dideoxy (dd)-GTP produces an extension product eight nucleotides longer if the cytidine has not been edited (CAA in the Figures); that is, incorporation of ddGTP causes chain tennination. If editing has created a uridine, then primer extension continues a further 11 nucleotides to the next 5' cytidine, where chain termination then occurs (UAA in the Figures).
  • Quantification of the level of editing is accurately determined using laser scanning densitometry.
  • the linear exposure range of the Phosphorlmager screen is sufficiently great to permit precise determination of low counts in the UAA bands whilst the high levels of counts in the CAA band remain in the linear range.
  • Editing percentages were calculated as the counts in the UAA band divided by the total counts in the CAA plus UAA bands times 100.
  • This assay has a lower level of detection of 0.1 %editing and remains linear up to 99.5%and is independent, between 1 ng and 500 ng, of the total amount of template PCR product used (M. P. Sowden, unpublished work). 217. Rev complementation/editing assays (Taagepera, S., et al. (1998) Proc.
  • transactivator DNA 0.75 ⁇ g of transactivator DNA (pRc/CMV vector or a nucleocytoplasmic shuttling competent Rev-Rex fusion; a gift of Dr Thomas J. Hope, Infectious Disease Laboratory, Salk Institute for Biological Studies, La Jolla, CA, U.S.A.) and 0.25 ⁇ g of pRS V-p--galactosidase [internal confrol for chloramphenicol acetyl-fransferase (CAT) assays] were introduced into McArdle cells using lipofectAMINE ® as described above.
  • the apoB pre-mRNA reporter constract contained an abbreviated splicing cassette from the adenovirus late leader sequence fused to 450 nt of wild-type apoB mRNA ( Figure 1A). Unspliced pre-mRNA and spliced mRNA were amplified from total cellular McArdle cell mRNA using the MS1/MS2 and SP6/T7 amplimer pairs respectively ( Figure 1A).
  • GenBank2 for apoB mooring-sequence similarities reveals numerous potential editing sites. However, many are located short distances from splice sites or within 5' or 3' untranslated regions or introns where the functional consequence(s) of a cytidine-to-uridine editing event is unclear. The release of the
  • Pre-mRNA transcripts were amplified using the Exl/Ex2 amplimers followed by nested PCR with the MS ⁇ 5/MS ⁇ 6 amplimer pair and were edited at an efficiency of 0.4 % ( Figure 3B).
  • Intron-containing transcripts were amplified using the Attorney Docket Number 21108.0035P1
  • MS ⁇ 5/MS ⁇ 6 amplimers followed by nested PCR with the MS2/MS3 amplimer pair and were edited at an efficiency of 0.5 % ( Figure 3B).
  • the use of the MS ⁇ 5/MS ⁇ 6 amplimer pair in the initial PCR would not distinguish between unspliced pre-mRNA or spliced-out lariat RNA, but given the rapid degradation of lariat RNA, it is unlikely that the amplified PCR products represent lariat RNA species. If, however, there were amplified lariat species present, the difference of 0.1 % between infron-containing and unspliced pre-mRNA suggests that lariat RNAs containing apoB editing sites are not efficient editing substrates.
  • Wild-type apoB cDNA transcripts expressed in wild-type McArdle cells edit 2-3-fold more efficiently than the endogenous franscript (Sowden, M., et al. (1996) RNA 2, 274-288.; Sowden M.P., et al. (1998) Nucleic Acids Res. 26:1644-1652.). It has been demonstrated that chimeric splicing-editing reporter RNAs (EVS-apoB) had low editing efficiency as nuclear transcripts, which did not change once spliced mRNAs had entered the cytoplasm ( Figure 1; (Yang, Y., et al. (2000) J. Biol. Chem. 275: 22663-22669.)). Hence the window of opportunity for a transcript to be edited in wild-type cells was confined to the nucleus, and when introns are proximal to the editing site, its utilization was impaired. Attorney Docket Number 21108.0035P1
  • RNA splicing and RNA nuclear export were separated by utilizing a modification of the Rev complementation assay that has been employed to identify HEV-l Rev-like nuclear export sequences (Taagepera, S., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:7457- 7462.). Rev functions, by interaction with an RRE, to export unspliced RNA out of the nucleus.
  • a reporter plasmid was constructed which contained an infron interrupted by the CAT gene and a functional apoB RNA editing cassette (Figure 4A).
  • CAT activity could only be expressed if unspliced RNA was exported to the cytoplasm, a process wholly dependent upon an active Rev protein expressed from a co-fransfected plasmid. In the presence of Rev, spliceosome assembly on the transcript does not occur and therefore should not interfere with the utilization of the apoB editing site contained with the intron. 228.
  • McArdle cells were co-fransfected with the modified reporter construct, together with either a control vector or a Rev expression vector. CAT activity was determined 48 h later ( Figure 4B).
  • the components of 5 the minimal editosome from defined in vitro system analyses are APOBEC-1 as a homodimeric cytidine deaminase (Lau, P.P., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91 :8522-8526.) bound to the auxiliary protein ACF/ASP that serves as the editing-site recognition factor tlirough its mooring-sequence-selective RNA-binding activity (Mehta, A., et al. (2000) Mol. Cell. Biol. 20:1846-1854; Lellek, H., et al. (2000) J. Biol.
  • RNA editing is communicating a temporal and spatial relationship that ultimately determines the efficiency of mooring-sequence-dependent editing. Consistent with this communication between the spliceosome and editosome is the finding that several proteins that have a role in RNA structure and/or splicing have also been implicated in RNA editing as auxiliary factors. These include hnRNP C, hnRNP D, APOBEC- 1 -binding protein (which has homology with hnRNP A and B) and KSRP, a protein involved in alternative splice site utilization (Lellek, H., et al. (2000) J. Biol. Chem. 275:19848- 19856; Greeve, J., et al. (1998) J. Biol. Chem. 379:1063-1073; Anant, S.G., et al.
  • RNA splicing and editing The description of the relationship of RNA splicing and editing is unique for apoB cytidine-to-uridine mRNA editing.
  • an emerging theme in RNA processing is an interdependence of multiple steps in RNA maturation.
  • Perhaps the most relevant to apoB editing is the adenine-to-inosine editing of glutamate and 5- hydroxytryptamine receptors.
  • mRNA substrates that undergo adenine-to-inosine editing all require the presence of a complementary intron sequence to form a partially double-stranded RNA structure that is recognized by the appropriate ADAR1 or ADAR2 enzyme Simpson, L., et al. (1996) Annu. Re.
  • GluR2 mRNA from neuronal cell lines is always edited to 100% at the Gln/Arg site, whereas unspliced GluR2 franscripts are edited to only 70-90% (Higuchi, M., et al. (1993) Cell. 75:1361-1370.), indicating a partial inhibition of splicing until editing has occuned.
  • the franscript of the Glu- 5 R6 gene contains three exonic editing sites (Ile/Val, Tyr/Cys and Gln/Arg) which are edited to different extents, indicating that there must be a tightly regulated and coordinated action of the appropriate ADAR and the spliceosome at each editing site (Kohler, M., et al. (1993) Neuron 10:491-500; Seeburg, P.H., et al. (1998) Brain Res. Rev. 26:217-229.). In crosses of ADAR2 +/- with GluR-B (R) +/+ mice, an influence
  • RNA polymerase H 15 subunit of the RNA polymerase H. This represents an efficient process for increasing local concentrations of related processing and transcription factors on pre-mRNAs as and when they are needed (Lewis, J.D., et al. (2000) Science 288:1385-1389.). Many analyses of RNA processing have attempted to identify active versus inactive populations of processing factors and have postulated that the greatest concentration of 0 factors may or may not conespond to sites of function, dependent upon metabolic activity (Spector, D. (1993) Annu. Rev. Cell. Biol. 9:265-315.). Specifically, recent photobleaching studies (Lewis, J.D., et al. (2000) Science 288:1385-1389.
  • RNAs that contain nonsense codons and that have passed through the spliceosome are 'marked' and targeted for decay (Le Hir, H., et al. (2000) EMBO J. 19:6860-6869.).
  • This imprinting of nuclear pre-mRNA by proteins that remain bound in the cytoplasm is a means of mRNAs 'communicating their history' (Kataoka, N., et al. (2000) Mol. Cell. 6:673-682.) and/or perhaps ensuring that no further RNA processing/editing occurs in the cytoplasm (Maquat, L., et al.
  • the infectivity assay was carried out in the context of Vif minus pseudotyped virases and 293 T cells either lacking or expressing CEM15.
  • An assay was developed using VSV G-protein pseudotyped lentiviral particles that confirmed the inhibitory effect of CEMl 5 on the infectivity of vif+ and vif- HEV-l particles and is amenable to the rapid demarcation of the regions of HEV-l DNA (or RNA) that is the target for CEMl 5 catalytic activity.
  • HEV-l proviral DNA vector (derived from pNL43; AEDS Reagent Repository) was modified by replacement of Nef with a GFP reporter gene and two in-frame stop codons were inserted that abolished vif production (pHR-GFP ⁇ Vif) (confirmed by western blotting with anti- Attorney Docket Number 21108.0035P1
  • Vif antibodies (AEDs Reagent Repository). Stable, HA-tagged CEMl 5 expressing 293T cell lines were selected with puromycin and verified by western blotting with a HA specific monoclonal antibody (HA.11; BabCo) ( Figure 11). The expression of similar levels of full-length HA-tagged CEMl 5 (or mutant derivative thereof) can be assayed as well. Although stractural modeling can predict focused mutations that impair deaminase activity without destabilizing the entire protein, expression of the mutants should be verified. The addition of the HA epitope tag has no effect on the ability of CEM15 to suppress infectivity (Sheehy et al. Nature 418:646-650, 2002).
  • HEV-l pro-viral DNAs will be packaged into pseudotyped lentiviral particles by co-transfection with a plasmid encoding the VS V G-protein into 293T cells that lack endogenous CEM15 (-) or expressed wild type CEM15 (+) ( Figure 11).
  • the resulting pseudotyped particles contain HEV-l RNA of near full-length (with only a ⁇ 2kb deletion) were quantified by reverse transcriptase (RT) assay.
  • RT reverse transcriptase
  • p24Gag protein content can also be assayed by ELISA to normalize viral particles. A defined number (lxlO 5 cpm of RT activity) of these particles were added to target, viras susceptible
  • the cellular deaminase CEMl 5 can introduce multiple and therefore catastrophic dC to dU mutations in negative sfrand viral DNA following reverse transcription.
  • This anti-viral activity is due to the inherent catalytic activity of CEMl 5 on single sfranded DNA and requires assembly of CEMl 5 within virions such that it is in position to interact with nascent cDNA during viral replication in the early stages of the HEV-l life cycle.
  • Antiviral activity of CEMl 5, however, can be blocked by the viral .
  • Vif viral infectivity factor
  • Vif interacts with CEMl 5 and induces its poly-ubiquitination and degradation through the proteosome, thereby reducing the abundance of CEMl 5 and
  • Changes in viral infectivity can be determined by ELIS A quantification of HEV p24 antigen released from CEMl 5 positive cells that have been infected with wild type HEV-l and treated with or without peptide Vif antagonists.
  • Western blotting for CEMl 5 can be conelated with peptide Vif antagonist protection of CEMl 5 with VDA suppression of viral infectivity.
  • APOBEC-3B (h3B), previously refened to as Phorbolins, (Jarmuz et al., Genomics, 79(3):285-96 (2002)) are co-expressed in human lymphoid and myeloid cells, and as is the case for APOBEC-1, can form homodimers and also heterodimers (Bogerd et al., Proc Natl Acad Sci U S A 101(11):3770-4 (2004)). It has been shown that CEM15 and APOBEC-3F deaminate deoxycytidine on HEV-l and HEV-2 minus 0 strand cDNA.
  • APOBEC-3F and APOBEC-3B establish a close proximity with viral genomes, by becoming integrated within virions during their assembly (Stopak et al., Mol Cell, 12:591-601 (2003); Gaddis et al., J. Virol, 77(10):5810-5820 (2003); Mariani et al., Cell. 114(1):21-31 (2003); Wiegand, et al., Embo J, 23(12):2451-8 (2004)).
  • dimers of deaminases such as APOBEC-1 and AJD are predicted to contain two catalytic centers (Xie et al., Proc Natl Acad Sci U S A, 101(21): 8114-9 (2004)). From structural modeling, it appears that in the dimer, a flexible flap domain from one catalytic center interacts with the other catalytic center and thereby regulates nucleic acid subsfrate binding.
  • CEMl 5, APOBEC-3F and APOBEC-3B monomers each have two catalytic centers (both of which have activity (Mangeat et al., Nature, 424(6944): 99- 103 (2003); Shindo et al., J Biol Chem, (2003)).
  • Homo- and heterodimers of CEMl 5, APOBEC-3F and APOBEC-3B therefore are predicted to have four catalytic centers and are likely to have considerable combinatorial substrate targeting potential that provides the host cell with an adaptive advantage against a broad spectrum of viruses.
  • HEV-l and HIV-2 use Vif to defeat the deaminase host defense.
  • Vif has been shown to bind to both CEMl 5 and APOBEC-3F to target their ubiquitination and proteolytic degradation via the proteosome (Stopak et al. (2003); Mariani et al. (2003); Yu, X., et al. Science 302(5647): 1056-60 (2003); Zheng et al. J Virol. 78(l l):6073-6 (2004)).
  • Vif s interaction with CEMl 5 occurs in a noncatalytic region that lies C- terminal to first catalytic domain.
  • a single amino acid within this region provides the essential charge for the interaction of CEMl 5 with Vif (Bogerd et al. (2004); Mariani et al (2003), and Wiegand (2004)).
  • Site-directed mutagenesis has shown that this single amino acid change in an ARP alters host range of a retroviruses (Bogerd et al. (2004), Mariani et al. (2003) and Xu et al., Proc Natl Acad Sci U S A, 101(15):5652-7 (2004).
  • SEV simian viras
  • Vif forms homodimers, and Vif dimerization is required for viral infectivity. It has also been shown that Vif dimerization is required for Vif-dependent destruction of CEMl 5. Therefore, the Vif dimerization domain is a drug target for suppressing viral infectivity. HEV is notorious for its hypermutability and the acquired
  • CEMl 5 abundance can be quantified by western blotting as described above. Small molecules that bind to any of the aforementioned peptides can be evaluated for their ability to protect or restore CEMl 5 abundance using the aforementioned western blotting systems of whole cell extracts of cells that have been
  • Vif dimerization domain Small molecules that bind to the Vif dimerization domain and evaluate their ability to block Vif dimerization, prevent CEMl 5 degradation and suppression HEV-l infectivity. Peptides conesponding to the Vif dimerization can be used to screen chemical libraries for interacting compounds. 5 248. Analysis of the initial hits. The screen can yield numerous compounds.
  • the initial evaluations can be done based on their ability to restore CEMl 5 abundance in Vif expressing cells using the western blotting assay described previously. This assay was chosen for the initial analysis of compounds over infectivity assays because given that CEMl 5
  • the screening nanows the pool of selected candidates from the initial screen to a half dozen or less compounds (SMVA candidates) for further validation.
  • SMVA candidates half dozen or less compounds
  • SMVA candidates then move on to secondary biological end point evaluations. This involves analysis of their ability to supress live virus infectivity as described above. Dose response curves can be established for all compounds that block viral infectivity.
  • CEMl 5 (a.k.a. APOBEC-3G or h3G) functions as a natural defense against HEV-l viral infectivity by mutating the viral genome during its reverse
  • HEV-l viral infectivity factor Vif
  • HEV-l viral infectivity factor Vif
  • CEM15 expression induced a marked (100-fold) reduction in viral particle production in the absence of Vif compared to that obtained from confrol cells or in the presence of Vif. This effect was due to a selective and marked reduction in viral protein and RNA.
  • CEMl 5 can bind directly to RNA, which shows that it can play a role in the reduction of viral RNA.
  • the phenotype described here differs from that in other reports in that it does not require CEMl 5 to become incorporated within virions or have mutagenic activity during reverse transcription. This mechanism can contribute important antiviral activity during late stages of the viral life cycle.
  • Reverse transcription-dependent mutational activity of CEM15 on HEV- 1 ssDNA is not the only means by which CEM 15 can reduce viral infectivity.
  • mutations in one or both of the zinc-dependent cytidine deaminase domains did not ablate CEM15's antiviral activity (Shindo et al., J Biol Chem (2003)).
  • blockage of reverse transcriptase (RT) processivity by CEMl 5 binding to the viral RNA templates has been suggested as an additional antiviral mechanism (Li et al., J Cell Biochem 92, 560-572 (2004)).
  • transient expression of CEM 15 reduced the level of pseudotyped HEV-l particles generated from producer cells that were co- transfected with replication-defective proviral DNA constructs and helper plasmids (Sheehy et al., Nature 418, 646- 650 (2002)). This antiviral activity would have had to involve a mechanism that was independent of reverse transcription.
  • CEMl 5 cDNA was RT-PCR amplified from oligo(dT)- primed total cellular RNA from CEM cells (Sheehy et al (2002).
  • CEMl 5 deaminase domain mutations (DM) [E67A, E259A] were created by site-directed mutagenesis using the Quikchange system (Stratagene). Wild type CEMl 5 and DM were subcloned with an amino-terminal 6xHis and HA (hemagglutinin) tag into pfRES- P to permit CMV promoter driven expression of the cDNA and puromycin selection from an ECMV IRES element.
  • pDHTV-GFP from Dr. V.
  • Planelles is a pNL4-3 derived HIV-1 vector that contains a deletion of the env gene.
  • pDHEV-GFP/ ⁇ Vif was constructed by inserting a 12bp fragment (5'-TAGTAACCCGGG-3', SEQ ID NO: 62) containing two termination codons underlined) at the PflMl site of pDHEV-GFP that lies near residue 89 of Vif, thereby leading to the production of a truncated and nonfunctional vif gene product.
  • Cell culture and Transfection- 293T cells obtained from ATCC were maintained in DMEM containing 10% fetal bovine serum plus penicillin/streptomycin/fungizone (Cellgro), and Non-Essential Amino Acids (Invitrogen) and were transfected using FuGENE 6 (Roche Molecular Biochemicals).
  • Clonal cell lines were obtained by limiting dilution under 1 ⁇ g/ml puromycin selection.
  • Virus production A two plasmid system was used to generate pseudotyped HEV-l particles. 293T cells stably expressing CEM15, DM, or empty pERES-P vector were fransfected with a mixture of pVSV-G and pDHEV-GFP (wt Vif) or pDHEV-GFP/ ⁇ Vif using Lipofectamine 2000 (Invitrogen). Viruses were harvested at 48 and 72 hour post- transfection from culture supernatants and concentrated by ulfracentrifugation (22 K rpm,2 hour at 4oC). Attorney Docket Number 21108.0035P1
  • Cell lysates and western blot analysis Cells were harvested by scraping into PBS containing a cocktail of protease inhibitors (0.5 ⁇ g/mL each of aprotinin, pepstatin, and leupeptin, 1 mM PMSF (USB Corp), 2 mM Benzamidine and 2 mM
  • RNA binding reactions were 5 incubated at 30 oC for 3 h as previously described (Smith (1998)).
  • the probe was GP-RNA cDNA radiolabeled with 3 2 P[dCTP] using Ready-To-Go DNA labeling beads (Amersham Biosciences) according to the manufacturer's protocol. Blots were hybridized to the probe (lxlO 6 Attorney Docket Number 21 108.0035P1
  • Blots were then stripped and reprobed with adenovirus E1A cDNA radiolabeled with 32 P[dCTP] as stated above.
  • 293T cell lines stably expressing CEMl 5 (293T-CEM15) were selected and fransfected with plasmids containing replication-defective (Env- deleted) HEV-l proviruses (Vif+ or ⁇ Vif) plus a helper/packaging plasmid (encoding
  • VSV-G Culture supernatants from these cells were then assayed by p24 ELIS A, and a marked reduction of viral particle production (100-fold) by the ⁇ Vif construct was detected in 293T- CEMl 5 versus the confrol, a 293T stable cell line containing pERES- P vector (Fig. 14). In contrast, Vif+ pro virus culture supernatants contained abundant viral particles, only 5- fold below control cells (Fig. 14). The infectivity of the
  • CEM 15 is predicted to contain two zinc-dependent deaminase domains (Wedekind et al. Trends Genet 19:207-216 (2003)), each of which has been shown to possess partial antiviral activity (Shindo et al. (2003)). Point mutations of the essential glutamate residue within each catalytic domain reduced significantly, but did not
  • proviral DNA isolated from 293T-CEM15 cells and control cells. No difference in DNA recovery was detected in 293T-CEM15 fransfected with ⁇ Vif provirus compared to control cells transfected with 4-Vif provirus, and no dC to dU mutations in proviral DNA were evident as determined by uracil DNA glycosylase treatment of isolated viral
  • CEMl 5 might have the ability to selectively target the frameshift region in the viral Gag-Pol mRNA. This was of
  • CEMl 5 RNA binding capacity was determined in- vitro using purified recombinant CEMl 5 and radiolabeled RNA in our standardized ultraviolet light (UV) crosslinking assay (Smith, H.D. (1998), Galloway et al. 34: 24-526, 528, 530 (2003)). CEMl 5 bound to radiolabeled HEV-l GP-RNA in concenfration dependent manner
  • Fig. 16C 20 proviras
  • CEMl 5 expression did not affect the abundance of an endogenous transcript present in 293 T cells (adeno viras El A RNA), as expected since luciferase and ⁇ -actin protein expression were also unaffected by CEMl 5.
  • El A RNA served as an internal loading control for comparison of viral RNA levels (Fig. 16C).
  • Expression of the deaminase inactive DM also induced a depletion of viral RNA but to
  • CEM 15 suppressed HEV-l production, which does not depend on the incorporation of CEM 15 into the virion and/or viral reverse transcription. It was shown that CEM 15 selectively reduced viral RNA and protein abundance resulting in a phenotype of reduced viral particle assembly. This effect was not dependent upon CEM15-mediated DNA mutation or RNA editing and was largely abrogated by the expression of Vif. It ; was also revealed that recombinant CEM 15 can bind directly to viral Gag-Pol RNA and • non- viral RNAs.
  • CEM 15 binding to viral RNA can lead to its premature decay, hi this regard, CEMl 5 interactions with Gag nucleocapsid (Cen et al. J Biol Chem (2004), Alee et al. J Biol Chem (2004)), and the ability of both proteins to bind HEV-l RNA can provide specificity resulting in the selective degradation of viral RNAs.
  • CEM 15 on viral RNA stability and protein production is therefore attributed to the fact that stable cell clones were used that uniformly express CEMl 5.
  • CEM 15 expression had a differential effect on viral protein abundance.
  • the expression of the 55 kDa Gag precursor (p55) in proviral fransfected 293T-CEM15 cells was similar, regardless of whether Vif was expressed, but p24 abundance was markedly reduced in the absence of Vif (Fig. 15 A).
  • the elevated levels of the ⁇ 55 in 293T-CEM15 cells and DM cells fransfected with D Vif provirus, compared to confrol cells and DM cells fransfected with 4-Vif provirus (where p55 undergoes rapid and efficient cleavage) throughout the 72 hours suggested a lack of protease activity (compare Fig. 15A and B -Vif, contrast to B +Vif and C).
  • CEMl 5 can exert an antiviral effect during both the early and late phases of the HEV-l life cycle.
  • HEV-l reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. Embo J. 8(11):3279- 85 (1989).
  • GRY-RBP as an apo B mRNA binding protein that interacts with both apobec-1 and with apobec-1 complementation factor (ACF) to modulate C to U editing. J. Biol. Chem. 276(13): 10272-10283 (2001).
  • Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in- frame stop codon. Science 238, 363-366 (1987).
  • Gerber, A, H. Grosjean, T. Melcher, and W. Keller Tadlp a yeast iRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADARl and ADAR2. Embo J. 17(16):4780-9 (1998).
  • RNA editing enzyme APOBECl and some of its homologs can act as DNA mutators.” Mol Cell 10(5): 1247-53.
  • RNA editing enzyme APOBECl and some of its homologs can act as DNA mutators. Mol Cell. 10(5): 1247-53 (2002).
  • HEV human immunodeficiency viras
  • RNA editing of AMP A receptor subunit GluR-B a base-paired intron-exon structure determines position and efficiency. Cell 75:1361-1370 (1993).

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Abstract

Disclosed are compounds that enhance RNA or DNA editing, as well as methods of using, identifying, and making such compounds. These compounds include Vif antagonists and cytidine deaminase inhibitors.

Description

Attorney Docket Number 21 108.0035P1
CYTIDINE DEAMINASE ACTIVATORS, DEOXYCYTIDINE
DEAMINASE ACTIVATORS, VTF ANTAGONISTS, AND METHODS OF SCREENING FOR MOLECULES THEREOF
This invention claims priority to United States Provisional Application No. 5 60/499,953, filed September 3, 2003.
This invention was made with government support under Grants RR15934, DK43738-08, F49620-01, and AI058789 from the National Institutes of Health. The government has certain rights in the invention.
I. BACKGROUND OF THE INVENTION
10 1. HIV- 1 , a human lentivirus, is the causative agent of AIDS, which presently infects approximately 42 million persons worldwide with 1 million infected persons in North America (http://www.unaids.org). The high mutation rate of HIV- 1 has in the past made it impossible to develop therapies that retain their effectiveness. Cunent therapies for HIV infected patients target the production of new viras by antiviral agents
15 that prevent replication of the viral RNA genomes into DNA prior to integration of the HIV DNA into chromosomal DNA or the disruption of the production or function of viral encoded proteins that are necessary for production of infectious viral particles. Antiviral agents that target viral replication have blunted the course of disease in patients already infected with HJV but these drags have side effects due to toxicity and,
20 while extending life for many patients, ultimately fail. Disruption of viral encoded protein production has not been as effective due largely to the high mutation rate of HEV and the consequent changing of viral protein into forms that retain function but no longer provide specific targets for the therapy. A combination of therapies together with better screening of blood supplies and blood products, improved public education
25 and safe-sex practices has curbed the spread of disease only in developed countries but, even in these countries, preventative measures exhibit incomplete confrol over the spread of the virus.
2. Human white blood cells express a protein called CEM15, a cytidine deaminase, which can change the genetic code of the infecting AEDS viruses. These
30 changes can render the virus incapable of producing an infection when they occur in critical genes encoding viral proteins and/or when they occur extensively throughout the HEV-1 genome. The AEDS viras, however, expresses a protein called Viral infectivity Attorney Docket Number 21108.0035P1
factor (Vif) that impairs the ability of CEM15 to act on viral DNA. Interrupting deaminase functions in other systems such as the apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1) and Activation Induced Deaminase (AED) systems have similar significance in the treatment of other diseases such as hypercholesterolemia and Hyper-IgM syndrome and certain forms of cancer (i.e., colorectal, APOBEC-1 and various leukemias and lymphomas). Thus, needed in the art is a means of enhancing deaminase function, or in the case of cancers, reducing or eliminating activity.
II. SUMMARY OF THE INVENTION 3. hi accordance with the purposes of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to Vif antagonists,. This invention also relates to cytidine deaminase activators, CEM15 activators, APOBEC-1 activators, and AED activators, and methods of identifying and making such agents.
4. In another aspect, this invention relates to deoxycytidine deaminase activators, ARP activators, and methods of identifying and making such activators.
5. Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
IILBRIEF DESCRIPTION OF THE DRAWINGS 6. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.
7. Figure 1 shows the effect of introns on editing efficiency. (A) Diagram of the chimeric apoB expression constructs. The intron sequence (EVS) is derived from the adeno viras late leader sequence. Co-ordinates of the human apoB sequence are shown and the location of PCR amplimers indicated. X indicates the deleted 5' splice donor or 3' splice acceptor sequences. CMV is cytomegalo virus. (B) Poisoned-primer-extension Attorney Docket Number 21108.0035P1
assays of amplified apoB RNAs. Pre-mRNA and mRNA were amplified with the MS1/MS2 or SP6/T7 amplimers respectively. Editing efficiencies, an average for triplicate transfections, for each RNA are shown beneath. Percent editing efficiency was determined as the number of counts in edited apoB mRNA (UAA) divided by the sum of counts in UAA plus those in unedited apoB mRNA (CAA) and multiplied by 100.
8. Figure 2 shows the effect of intron proximity on editing efficiency. (A) Diagram of the chimeric apoB expression constructs. EVS-(EVSΔ3'5')-apoB and EVS- (IVSΔ3'5') -apoB were created by the insertion of one or two copies respectively of the EVSΔ3'5' intron cassette into EVS-apoB. Human apoB co-ordinates and amplimer annealing sites are indicated (Figure 1). (B) Poisoned-primer-extension assays of amplified apoB RNAs. Pre-mRNA and mRNA were amplified with the MS7/MS2 or SP6/T7 amplimers respectively. Editing efficiencies, an average for duplicate transfections, for each RNA are shown beneath. 9. Figure 3 shows that the editing sites within introns are poorly utilized. (A)
Diagram of the chimeric apoB expression constructs. The apoB editing cassette was inserted as a PCR product into a unique HindlH site 5 'of the polypyrimidine tract in EVS-apoB and EVS-Δ3'5'apoB (Figure 1). Amplimer annealing sites are indicated. (B) Poisoned primer extension assays of amplified apoB RNAs. Unspliced pre-mRNA and infron containing RNA were amplified with the Exl/Ex2 or MS D5/MS D6 amplimers respectively. Editing efficiencies, an average for duplicate transfections, for each RNA are shown beneath.
10. Figure 4 shows that editing is regulated by RNA splicing. (A) Diagram of the modified CAT reporter construct (CMV128) used in the Rev complementation assay; a gift from Dr Thomas J. Hope of the Salk Institute. The splice donor (SD), splice acceptor (SA), RRE, intron and 3' long tandem repeat (LTR) are from the HEV-1 genome. CMV128 was modified by insertion of the apoB editing cassette as a PCR product into the BamHI site 3' of the CAT gene. Amplimer annealing sites are indicated. (B) McArdle cell CAT activity in the absence (Vector) or presence of the Rev transactivator. Values are averages for duplicate experiments. CMVCAT was an assay confrol transfection. (C) Poisoned-primer-extension assays of amplified apoB Attorney Docket Number 2110S.0035P1
RNAs. 'hitron and exon RNA' was amplified using the EF MS2 amplimers. Editing efficiencies for each RNA are shown beneath. Promiscuous editing is indicated by ' 1 '. 11. Figure 5 shows representative members of the APOBEC-1 related family of cytidine deaminases including CEM15. Also are APOBEC-1 complementation factor (ACF) and viral infectivity factor (Vif). The catalytic domain of APOBEC-1 is characterized by a ZDD with three zinc ligands (either His or Cys), a glutamic acid, a proline residue and a conserved primary sequence spacing (Mian, I.S., et al, (1998) J Comput Biol. 5:57-72.). The ZDD of other deaminases and APOBEC-1 related proteins is shown for comparison along with a consensus ZDD. The indicated residues in the catalytic site of APOBEC-1 bind AU-rich RNA with weak affinity. The leucine rich region (LRR) of APOBEC-1 has been implicated in APOBEC-1 dimerization and shown to be required for editing (Lau, P.P., et al, (1994) Proc Natl Acad Sci U S A. 91:8522-6; Oka, K., et al, (1991) J Biol Chem. 272:1456-60.) but structural modeling suggests that LRR forms the hydrophobic core of the protein monomer (Navaratnam, N., et al, (1998) J Mol Biol. 275:695-714.). ACF complements APOBEC-1 through its APOBEC-1 and RNA bindings activities. The RNA recognition motifs (RRM)s are required for mooring sequence-specific RNA binding and these domains plus sequence flanking them are required for APOBEC-1 interaction and complementation (Blanc, V., et al, (2001) J Biol Chem. 276:46386-93.; Mehta, A., et al., (2002) RNA. 8:69-82.) APOBEC-1 complementation activity minimally depends on ACF binding to both APOBEC-1 and mooring sequence RNA. A broad APOBEC-1 complementation region is indicated that is inclusive of all regions implicated in this activity (Blanc, V., et al, (2001) J Biol Chem. 276:46386-93.; Mehta, A., et al, (2002) RNA. 8:69-82.) Experiments have shown the N-terminal half of Vif is necessary for viral infectivity (Henzler, T. 2001). However, reports have demonstrated that residues in the C-terminus (amino acids 151-164) are essential for infectivity (Yang, S. et al. 2001) and that multimerization of Vif through the motif PPLP within this region was essential for infectivity. Peptides capable of binding to this domain of Vif blocked Vif- Vif interactions and Vif-Hck interactions in vitro and suppressed viral infectivity in cell- based assay systems. Residues in the N-terminus of Vif are essential for RNA binding and packing of Vif within the virion (Zhang et al. 2000; Khan et al. 2001; Lake et al. 2003). Attorney Docket Number 21108.0035P 1
12. Figure 6 shows schematic depictions of the cytidine deaminase (CD A) polypeptide fold and structure-based alignments of APOBEC-1 with respect to its related proteins (ARPs). Figure 6a depicts a gene duplication model for cytidine deaminases. CDD1 belongs to the teframeric class of cytidine deaminases with a quaternary fold nearly identical to that of the teframeric cytidine deaminase from B. subtilis (Johansson, E., et al, (2002) Biochemistry. 41:2563-70.). Such teframeric enzymes exhibit the classical αββαβαββ topology of the Zinc Dependent Deaminase Domain (ZDD) observed first in the Catalytic Domain (CD) of the dimeric enzyme from E. coli (Betts, L., et al, (1994) J Mol Biol. 235:635-56). According to the gene duplication model, an ancesfral CDDl-like monomer (upper left ribbon) duplicated and fused to produce a bipartite monomer. Over time a C-terminal Pseudo-Catalytic Domain (PCD) arose that lost substrate and Zn2+ binding abilities (upper right ribbon). The model holds that the interdomain CD-PCD junction is joined via flexible linker that features conserved Gly residues necessary for catalytic activity on large polymeric DNA or RNA substrates. The function of the PCD is to stabilize the hydrophobic monomer core and to engage in auxiliary factor binding. The loss of PCD helix αl can provide a hydrophobic surface were auxiliary factors bind to facilitate substrate recognition thereby regulating catalysis. The enzymes remain oligomeric because each active site comprises multiple polypeptide chains. Modern representatives of the chimeric CDA fold include the enzyme from E. coli, as well as APOBEC-1 and AED. Other ARPs such as APOBEC-3G (CEM15) may have arisen through a second gene duplication to produce a pseudo-homodimer on a single polypeptide chain (lower ribbon); structural properties of the connector polypeptide are unknown. Signature sequences compiled from strict structure-based alignments (upper) are shown below respective ribbon diagrams, where X represents any amino acid. Linker regions (lines) and the location of Zn2+ binding (spheres) are depicted. Although experimental evidence suggests APOBEC-3B has reduced Zn binding and exists as a dimer (Jarmuz, A., et al, (2002) Genomics, 79:285-96), modeling studies suggest it will bind Zn2+ (as shown in Wedekind et al. Trends Genet, 19(4):207-16, 2003) and may function as a monomer. Inset spheres represent proper (222) CDDl-like quaternary structure symmetry whereas APOBEC-1 -like enzymes exhibit pseudo-222 symmetry relating CD and PCD subunits; in the latter enzyme a proper dyad axis relates the polypeptide Attorney Docket Number 21108.0035P1
chains. Finally, APOBEC-3G can fold as a monomer from a single polypeptide chain with each CD and PCD (differently colored spheres in lower left inset box) related by improper 222 symmetry with no strict axes of symmetry. Figure 6b depicts the stracture based sequence alignment for ARPs. Sequences from human APOBEC-1, 5 AED, and APOBEC-3G were aligned based upon a main-chain alpha-carbon least- squares superposition of the known cytidine deaminase three dimensional crystal stractures from E. coli, B. subtilis and S. cerevisiae (Figure 6c). Amino acid sequence alignments were optimized to minimize gaps in major secondary stracture elements, which are depicted as tubes (α-helices) and anows (β-strands) in Figure 6b.
10 Additionally, loops, turns, and insertions of Figure 6b are marked L and T and i, respectively. L-Cl and L-C2 represent distinct loop structures in the dimeric versus teframeric cytidine deaminases. Sections of basic residues that overlap the bipartite NLS of APOBEC-1 are marked BP-1 and BP-2. Figure 6d depicts a schematic diagram of the domain structure observed in APOBEC-1 and related ARPs based upon
15 computer-based sequence alignments using the ZDD signature sequence shown in the lower panel of Figure 6a.
13. Figure 7 shows the relation of CEM15 amino acid sequence to APOBEC-1 and other APOBEC-1 Related Proteins (ARPs) by use of standard computational methods based upon amino acid similarity or identity. Amino acid sequence alignments
20 illustrate conservation of Zn2+ ligands and key catalytic residues essential to the mechanism of hydroiytic deamination by cytidine deaminases (CD A). Collectively, these amino acids form a signature zinc-dependent deaminase domain (ZDD), present in: (i) APOBEC-1, which mediates C to U editing of apoB mRNA, (ii) the Activation Induced Deaminase (AID), which mediates Somatic Hypermutation (SHM) and Class
25 Switch Recombination (CSR), and (iii) CEM15, which blocks HIV-1 viral infectivity.
14. Figure 8 shows a schematic ribbon diagram depicting a three-dimensional model of APOBEC-1 derived from comparative modeling by the method of satisfying spatial restraints. Structure-based homology modeling has provided insight into the fold of APOBEC-1, and has been conoborated by protein engineering, site-directed
30 mutagenesis, and functional analyses. The cunent model for APOBEC-1 predicts a two domain stracture comprising a catalytic domain (CD) and a pseudo-catalytic domain (PCD) joined by a central linker, which folds over the active site (green segment). The Attorney Docket Number 21108.0035P1
linker sequence is conserved among ARPs (Figure 6b), and linker sequence composition and polypeptide chain length are essential for efficient RNA editing by APOBEC-1. The APOBEC-1 model also provides a rationale for losses in editing due to surface point mutations, such as F156L, located 25 A from the active site. This 5 aromatic to branched-chain hydrophobic change appears to have no influence on the stability of the enzyme core, but can be involved in auxiliary factor binding required for RNA binding. Similarly, a series of basic residues at BP2 (Figure 6b) are close to the active site, and can be responsible for RNA binding. Mutagenesis of all basic residues within the respective bp-clusters abolishes editing activity (Teng, 1999, J. Lipid Res.
10 40:623). The stractural template of the APOBEC-1 model is derived from the spatial constraints derived from a superposition of three high resolution CDA crystal stractures that exhibit a nearly identical αβ2αβαβ fold despite modest sequence identity (~24% Figure 6c); fold conservation also exists at the oligomeric level, since each enzyme exhibits either proper 222 or pseudo~222 symmetry. Similarities in the Zn2+ dependent
15 deaminase mechanism, as well as structural similarities among the known CD As of pyrimidine metabolism show that the ARP fold is evolutionarily conserved among dimeric CD As that act on RNA and DNA. Similarly, it is likely that CEM15 (APOBEC-3G) evolved from an APOBEC-1 -like precursor by gene duplication. Thus, the CEM15 stracture comprises two active sites per polypeptide chain with the
20 topology CD 1 -PCD 1 -connector-CD2-PCD2 (Figure 6a).
15. Figure 9 shows a structural model for CEM15. The use of comparative modeling by the method of satisfied spatial restraints has allowed the calculation of a CEM15 three-dimensional model including all atoms of the 384 amino sequence. Spatial restraints for the template were derived from the atomic coordinates of three
25 known CDA crystal structures including a bonafi.de RNA editing enzyme from yeast Cddl, which is capable of deaminating free nucleosides as well as polymeric RNA substrates, such as reporter apoB mRNA. The known CDA crystal structures represent both dimeric and teframeric quaternary folds (Figure 6a), which allows an accurate model to be prepared using multiple structural restraints. Further insight into the
30 CEM15 stracture has also been attained by analogy to modeling and functional results obtained from APOBEC-1. A comparative model of CEM15 was calculated by use of the program 'Modeller' and subsequently checked by the program suites PROCHECK Attorney Docket Number 21108.0035P 1
and the Verify3D server. The model was energy minimized using simulated annealing and molecular dynamics methods. No restraints were placed on secondary elements, except those derived from the triple CDA stracture alignment. The position of the UMP nucleotide was incorporated based upon spatial restraints derived from known 5 crystal structures. Zn atoms were restrained using reasonable coordination geometry derived from the known CD As. The resulting model demonstrated that the 384 amino acid sequence of CEM15 can be accommodated by a dimeric CDA quaternary fold (analogous to the E. coli CDA or APOBEC-1 with 2 x 236 amino acids).
16. Figure 10 shows possible CEM15 oligomers. The number of possible
10 CEM15 quaternary stractures is limited and the actual oligomeric state can be evaluated by gel filtration chromatography, or through site directed mutagenesis that evaluates the requirement of single or dual CD domains in CEM15 activity. For example, possible dimeric CEM15 stractures (Figures 10c and lOd) predict mutually exclusive intermolecular contacts with the distinguishing feature that the interaction observed in
15 Figure 10c is such that each CD pairs with itself, and similarly for each PCD. hi contrast, every domain in Figure lOd falls in a unique environment (i.e. no CD or PCD pairs with itself). A variety of truncation mutations address the question of whether or not a dimer of the form in head-to-head or head-to-tail exists in solution (Figure 10c versus lOd).
20 17. Figure 11 shows HA-tagged CEM15 in 293T cells. Stable, HA-tagged
CEM15 expressing 293T cell lines were selected with puromycin and verified by western blotting with a HA specific monoclonal antibody. The addition of the HA epitope tag has no effect on the ability of CEM15 to suppress infectivity. Isogenic HEV- 1 pro-viral DNAs are packaged into pseudotyped lentiviral particles by co-transfection
25 with a plasmid encoding the VSV G-protein into 293T cells that lack endogenous CEM15 (-) or expressed wild type CEM15 (+).
18. Figure 12 shows the results of the assay described in Example 4, indicating that the expression of CEM15 in 293T cells resulted in at least a 100-fold decrease in Vif- viral infectivity compared to particles generated in parental 293T cells. The low
30 level of GFP expression from vif-, CEM15+ particles is indistinguishable from background fluorescence in confrol cells. Attorney Docket Number 21108.0035P1
19. Figure 13 shows poisoned primer extension assays and Western analysis for Cddl mutants and chimeric proteins. In the context of late log phase growth in yeast with galactose feeding, overexpressed Cddl is capable of C to U specific editing of reporter apoB mRNA at site C6666 at a level of 6.7%, which is ~10x times greater than the negative control (Figure 13, empty vector - compare lanes 1 and 2). In confrast, the CDA from E. coli (equivalent to PDB entry 1AF2) is incapable of editing on the reporter substrate (Figure 13, lane 3). Similarly, the active site mutants E61A and G137A abolish detectable Cddl activity (Figure 13, lanes 4 and 5). Likewise, the addition of the E. coli linker sequence (Figure 13, lane 6) impairs editing function as well. In a series of chimeric constracts in which the Cddl tetramer was converted into a molecular dimer, the chimeric molecule appears functional, as long as an amino acid linker of 7-8 amino acids is used to join the respective Cddl subunits (Figure 13, Right Panel lanes 1-4). However, when the longer E. coli linker is used to join Cddl monomers, there is no detectable activity on the reporter substrate, although the chimeric protein is expressed (Figure 13, Western blot). Paradoxically, when conserved Gly residues of the APOBEC-1 linker (130 and 138) are mutated to Ala, the chimeric enzyme is still active (Figure 13, lanes 3 and 4 of right panel), although this result is consistent with the observation that APOBEC-3G can utilize a non-Gly residue at this position (Figure 6b). 20. Figure 14 shows reduced production of pseudotyped HIV-1 viral particles by cells expressing CEM15 or DM. p24 concentration (pg/ml) normalized to % GFP containing cells (as a measure of transfection efficiency) for 293T cells stably expressing pE ES-P vector (n=6), CEM15 (n=6) and DM (n=5), following transfection with wild-type (Vif+) or ΔVif pro viral DNA plasmids (black and white bars, respectively). Enor bars represent standard deviation calculated from n for each cell line.
21. Figure 15 shows CEM15 suppresses HIV-1 protein abundance. 293 T cell lines stably expressing (A) CEM15, (B) DM, and (C) control pLRES-P vector were transiently fransfected with proviral HEV-1 plasmids (containing either wild-type Vif (+) or ΔVif (-)). Total cell lysates were prepared at 24, 48, and 72 hours post-transfection, separated by SDS-PAGE and analyzed by immunoblot assay using antibodies reactive with HA (HA-tagged CEM15 and DM), Vif, p24, RT, β- Attorney Docket Number 21108.0035P1
actin, Vpr, or Tat (as denoted on the left). The molecular weight (kDa) of the indicated protein species is given to the right.
22. Figure 16 shows CEM15 suppresses HEV-l viral RNA abundance. (A) Location of Gag-Pol junction and protease region of HEV-l genomic RNA conesponding to the GP-RNA probe used for RNA binding and northern blot analysis. (B) UV crosslinking of increasing concentration of recombinant CEM15 protein (1, 2 and 4 μg protein) to 20 frnol radiolabeled GP-RNA and apoB RNA. (C) Poly A+ RNA abundance for Gag-Pol transcripts in 293T-CEM15 at 24, 48, and 72 hours and DM cells at 48 hours post- transfection with Vif+ (black) and ΔVif (white) proviral DNA. Results are expressed as the ratio of viral RNA (GP-RNA region) to endogenous cellular RNA (adeno virus El A) determined through phosphorimager scanning densitometry analysis of northern blots.
IV. DETAILED DESCRIPTION 23. The invention provides compounds that enhance RNA or DNA editing, as well as methods of using, identifying, and making such compounds. The compounds are useful in preventing or treating a variety of diseases, including viral infections. Described herein are cytosine deaminase activators and antagonists of compounds, like viral infectivity factor (vif), that interfere with deaminases. A. RNA and DNA editing 24. There are several examples of cellular and viral mRNA editing in mammalian cells. (Grosjean and Benne (1998); Smith et al. (1997) RNA 3: 1105-23). Two examples of such editing mechanisms are the adenosine to inosine and cytidine to uridine conversions. (Grosjean and Benne (1998); Smith et al. (1996) Trends in Genetics 12:418-24; Krough et al. (1994) J. Mol. Biol. 235:1501-31). Editing can also occur on both RNA and on DNA, and typically these functions are performed by different types of deaminases.
25. A to I editing involves a family of adenosine deaminases active on RNA (ADARs). ADARs typically have two or more double stranded RNA binding motifs (DRBM) in addition to a catalytic domain whose tertiary structure positions a histidine and two cysteines for zinc ion coordination and a glutamic acid residue as a proton donor. The catalytic domain is conserved at the level of secondary and tertiary structure among ADARs, cytidine nucleoside/nucleotide deaminases and CDARs but Attorney Docket Number 21108.0035P 1
differs markedly from that found in adenosine nucleoside/nucleotide deaminases (Higuchi et al (1993) Cell 75:1361-70). ADAR editing sites are found predominantly in exons and are characterized by RNA secondary structure encompassing the adenosine(s) to be edited. In human exon A to I editing, RNA secondary structure is formed between the exon and a 3 ' proximal sequence with the downstream infron
(Grosjean and Benne (1998); Smith et al. (1997) RNA 3: 1105-23; Smith et al. (1996) Trends in Genetics 12:418-24; Maas et al (1996) J. Biol. Chem. 271:12221-26; Reuter et al. (1999) Nature 399:75-80; O'Connell (1997) Current Biol. 7:R437-38). Consequently, A to I editing occurs prior to pre-mRNA splicing in the nucleus. The resultant inosine base pairs with cytosine and codons that have been edited, effectively have an A to G change. ADAR mRNA substrates frequently contain multiple A to I editing sites and each site is selectively edited by an ADAR, such as ADAR1 or ADAR2. ADARs typically function autonomously in editing mRNAs. ADARs bind secondary stracture at the editing site through their double stranded RNA binding motifs or DRBMs and perform hydrolytic deamination of adenosine through their catalytic domain.
1. APOBEC-1 26. One example of a Cytosine Deaminase Active on RNA (CDAR) is APOBEC-1 (apolipoprotein B mRNA editing catalytic subunit 1) (accession # NM_005889) encoded on human chromosome 12. (Grosjean and Benne (1998); Lau et al. (1994) PNAS 91:8522-26; Teng et al (1993) Science 260:1816-19). APOBEC- 1 edits apoB mRNA primarily at nucleotide 6666 (C6666) and to a lesser extent at C8702 (Powell et al. (1987) Cell 50:831-40; Chen et al. (1987) Science 238: 363-366; Smith (1993) Seminars in Cell Biology 4:267-78) in a zinc dependent fashion (Smith et al. (1997) RNA 3:1105-1123). This editing creates an in-frame translation stop codon, UAA, from a glutamine codon, CAA at position C6666 (Grosjean and Benne (1998); Powell et al. (1987) Cell 50:831-840; Chen et al. (1987) Science 238:363-66). The biomedical significance of apoB mRNA editing is that it results in increased production and secretion of B48 containing very low density lipoproteins and conespondingly, a decrease in the abundance of the atherogenic apoBlOO containing low density lipoproteins in serum (Davidson et al. (1988) JBC 262:13482-85; Baum et al. (1990) JBC 265:19263-70; Wu et al. (1990) JBC 265:12312-12316; Harris and Attorney Docket Number 21108.0035P1
Smith (1992) Biochem. Biophys. Res. Commun. 183:899-903; hiui et al. (1994) J. Lipid Res. 35:1477-89;Funahashi et al (1995) J. Lipid Res. 36:414-428; Giannoni et al. J. Lipid Res. 36:1664-75; Lau et al. (1995) J. Lipid Res. 36: 2069-78; Phung et al. (1996) Metabolism 45:1056-58; Van Mater et al. (1998)Biochem. Biophys. Res. 5 Commun. 252:334-39; von Wronski et al. (1998) Metab. Clin.Exp. 7:869-73). 27. In APOBEC-1 gene knockout mice, apoB mRNA was unedited, demonstrating that no other CDAR is expressed which can use apoB mRNA as a substrate (Nakamuta et al. (1996) JBC 271:25981-88;Morrison et al. (1996) PNAS 271:25981-88; Hirano et al. (1996) J. Biol. Chem. 271:9887-90; Yamanaka et al.
10 (1997) Genes Dev. 11 :321-33; Yamanaka et al. (1995) PNAS 92:9493-87; Sowden et al. (1998) Nucl. Acids Res. 26:1644-1652). ApoB is translated from a 14 kb mRNA that is transcribed from a single copy gene located on human chromosome 2 (Scott (1989) J. Mol. Med. 6:65-80). ApoB protein is a non-exchangeable structural component of chylomicrons and of very low density (VLDL) and low density (LDL)
15 lipoprotein particles. APOBEC-1 editing of apoB mRNA determines whether a small (apoB48) or a large (apoBlOO) variant of apoB lipoprotein is expressed (Grosjean and Benne (1998); Powell et al. (1987) Cell 50:831-840; Chen et al. (1987) Science 238:363-66; Scott (1989) J. Mol. Med. 6:63-80; Greeve et al (1993) J. Lipid Res. 34:1367-83).
20 28. In contrast to A to I editing, RNA secondary structure does not appear to be required for apoB RNA editing. Instead, apoB mRNA editing requires an 11 nucleotide motif known as the mooring sequence. Placement of the mooring sequence 4-8 nucleotides 3' of a cytidine within reporter RNAs is frequently sufficient for that RNA to support editing (Smith (1993) Seminars in Cell Biol. 4:267-78; Sowden et al.
25 (1998) Nucl. Acids Res. 26:1644-1652; Backus and Smith (1992) Nucl. Acids Res. 22:6007-14; Backus and Smith (1991) Nucl. Acids Res. 19:6781-86; Backus and Smith (1994) Biochim. Biophys. Acta 1217:65-73; Backus et al. (1994) Biochim. Biophys. Acta 1219:1-14; Sowden et al. (1996) RNA 2:274-88). The mooring sequence is left intact in edited mRNA and therefore its occunence downstream of a
30 cytidine is predictive of an editing site.
29. APOBEC-1 relies on auxiliary proteins for RNA recognition (Grosjean and Benne (1998); Teng et al. (1993) Science 260:1816-19; Sowden et al (1998) Attorney Docket Number 21108.0035P1
Nucl. Acids Res. 26:1644-52; Inui et al. (1994) J. Lipid Res. 35:1477-89; Dance et al. (2001) Nucl. Acids Res. 29:1772-80). APOBEC-1 only has weak RNA binding activity of low specificity (Anant et al. (1995) JBC 270:14768-75; MacGinnitie et al. (1995) JBC 270:14768-75). To edit apoB mRNA, APOBEC-1 requires a mooring 5 sequence-specific, RNA binding protein that binds apoB mRNA and to which
APOBEC-1 can bind and orient itself to C6666. Under defined in vitro conditions, apoB RNA, recombinant APOBEC-1 and proteins known as ACF/ASP (APOBEC-1 Complementing Factor/APOBEC-1 Stimulating Protein) were all that was required for editing activity and are therefore considered as the minimal editing complex or
10 editosome (Mehta et al. (2000) Mol. Cell Biol. 20:1846-54; Lellek et al. (2000) JBC 275:19848-56).
30. ACF was isolated and cloned using biochemical fractionation and yeast two hybrid genetic selection (Mehta et al. (2000) Mol. Cell Biol. 20:1846-54; Lellek et al. (2000) JBC 275:19848-56). Overexpression of 6His-tagged APOBEC-1 in
15 mammalian cells enabled the intracellular assembled editosome to be affinity purified (Yang et al. (1997) JBC 272:27700-06). These studies demonstrated that ACF associated with APOBEC-1 through 1M NaCl resistant interactions and that three other RNA binding proteins (100 kDa, 55 kDa and 44 kDa) with affinity for the mooring sequence co-purified with the editosome (Yang et al. (1997) JBC
20 272:27700-06). P100 and p55 were both mooring sequence selective RNA binding proteins but p44 was a general RNA binding protein. Additional studies utilizing yeast two hybrid analyses using APOBEC-1 affinity and antibodies developed against the editosome and ACF have demonstrated proteins such as hiiRNP ABBPl (Lau et al. (1997) JBC 272:1452-55), the alternative splicing factor KSRP (Lellek et al.
25 (2000) JBC 275: 19848-56) and alphaB (αI3) serum proteinase inhibitor as positive modulators of editing activity (Schock et al, (1996) PNAS 93:1097-1102) and hnRNP protein C (Greeve et al. (1998) Biol. Chem. 379:1063-73) and GRY-RBP (Blanc et al. (2001) JBC 276: 10272-83; Lau et al. (2001) Biochem. Biophys. Res. Commun. 282:977-83) as negative modulators of apoB mRNA editing.
30 31. Structure-based homology modeling has provided insight into the fold of
APOBEC-1 (Figure 8; Wedekind et al. Trends Genet, 19(4):207-16, 2003), and the modeling of APOBEC-1 has been conoborated by protein engineering, site-directed Attorney Docket Number 21108.0035P1
mutagenesis, and functional analyses. The cunent model for APOBEC-1 is a two domain structure comprising a catalytic domain (CD) and a pseudo-catalytic domain (PCD) joined by a central linker, which folds over the active site (Figure 8). The linker sequence is conserved among ARPs, and sequence identity and length are 5 essential for efficient RNA editing by APOBEC- 1. The APOBEC- 1 model also provides a rationale for losses in editing due to surface point mutations, such as F156L (Navaratnam et al. Cell 81(2)187-95), located 25 A from the active site. Such a change can influence auxiliary factor binding. Other mutations such as K33A K34A abolish activity (Teng et al. J Lipid Res, 40(4) 623-35, 1999). 10 32. Other mutations such as K33A/K34A abolish activity (Teng et al. J Lipid
Res, 40(4) 623-35, 1999). These basic residues are a feature of all ARP family members, including CDDl. In the model the latter basic residues are close to the active site, and can be responsible for RNA binding. The spatial restraints and fidelity of the APOBEC-1 model is derived from superposition of three high resolution CDA 15 crystal stractures (Betts et al. J Mol Biol 235(2):635-56, 1994; Johansson et al.
Biochemistry 41(8): p. 2563-70, 2000) that exhibit a nearly identical β αβαβ2 fold despite modest sequence identity (-24%); fold conservation also exists at the oligomeric level, since each enzyme exhibits proper or nearly proper 222 symmetry (Figure 6a and 6c). 20 33. Stractural homology is derived from the fact that dimeric CD As arose from gene duplication of a CD precursor (Betts et al. J Mol Biol 235(2):635-56, 1994; Johansson et al. Biochemistry 41(8): p. 2563-70, 2000) producing a PCD, which although catalytically inactive, forms an inextricable part of the core protein fold and the enzyme active site. Pairwise superpositions of 75 backbone atoms from the yeast 25 CDDl crystal structure with comparable atoms from those CDA structures of E. coli and B. subtilis results in rmsds of 1.22 A and 0.77 A, respectively (Figure 6c), which exceeds the stractural homology predicted by simple sequence alignments of proteins with unrelated function (Chothia et al. EMBO J. 5(4)823-6, 1986; Lesk et al. J Mol Biol, 136(3):225-70.). Notably the yeast enzyme CDDl, used in pyrimidine salvage, 30 edits ectopically expressed apoB mRNA in yeast. (Dance et al Nucleic Acids Res
29(8): 1772-80). Hence, it is conceivable that the CDA motif of nucleoside metabolism Attorney Docket Number 21108.0035P1
has been co-opted to function on larger RNA or DNA substrates due to variations at several stractural components including the active linker site.
34. Previously, the threading of the APOBEC-1 primary amino acid sequence onto the backbone atomic coordinates of the known crystal stracture of E. coli cytidine deaminase dimer indicated that APOBEC-1 structure was consistent with a head-to- head homodimer with the active CD domain of one monomer in apposition with the CD domain of the other monomer (Navaratnam et al. J Mol Biol, (1998) 275(4):695-714). In this model, one of two active deaminase domains is predicted to interact non- catalytically with a specific U from the RNA substrate while the other active domain interacts with the cytidine to be edited (Navaratnam et al. J Mol Biol, (1998)
275(4):695-714). Importantly, dimerization has been shown to be essential for editing activity (Lau et al. (1994) PNAS 91:8522-26; Navaratnam et al. (1995) Cell 81:187-95; Oka et al. (1997) JBC 272:1456-60). The model also predicted a leucine-rich region (LRR) in the C-terminus of APOBEC-1 as a functional motif characteristic of cytidine deaminases that function as dimers. The LRR is essential for APOBEC-1 homodimer formation, apoB mRNA editing, APOBEC-1 interaction with ACF, and APOBEC-1 subcellular distribution (Lau et al. (1994) PNAS 91:8522-26; MacGinnitie et al. (1995) JBC 270:14768-75; Navaratnam et al. (1995) Cell 81:187-95; Oka et al. (1997) JBC 272:1456-60). 2. AID
35. Other putative members of the CDAR family in humans were identified by genomic sequence analyses and include ADD (Muramatsu et al. (1999) JBC 274:18740-76; Muramatsu et al. (2000) Cell 102:553-564); Revy et al. (2000) Cell 102:565-76), APOBEC-2 (Liao et al. (1999) Biochem. Biophys. Res. Commun. 260:398-404) and variants of phorbolins, which are also known as the APOBEC3 family (Anant et al., (1998) Biol Chem. 379:1075-81; Jarmuz et al, (2002) Genomics. 79:285-96; Sheehy et al. (2002) Nature 418:646-50; Madsen et al. (1999) J. Invest. Dermatol. 113:162-69). These candidate CDARs have attracted interest because they share homology with the catalytic domain found in APOBEC-1 and the ADARs and they also have interesting physiological circumstances for their expression. One characteristic of the catalytic domain in CDARs and ADARs is the occunence and spacing of a histidine and two cysteines (or three cysteines), required for the Attorney Docket Number 21108.0035P1
coordination of a zinc atom, also known as the zinc binding domain or ZBD (Grosjean and Benne ((1998); Mian et al. (1998) J. Comput. Biol. 5:57-72). The ZBD of ADARs is distinguishable from that found in cytidine deaminases because the third cysteine in ADARs is located significantly further in primary sequence from the second conserved cysteine residue (Mian et al. (1998) J. Comput. Biol. 5:57-72;
Gerber et al. (2001) TIBS 26:376-84). The ZBD of APOBEC-1 is located in the N- terminal half of the protein and modeling has suggested that a pseudo- nonfunctional) ZBD domain is repeated in the C-terminus (Mian et al. (1998) J. Comput. Biol. 5:57-72). 36. Activation induced deaminase, AID (GenBank accession # BC006296) is encoded on human chromosome 12 (Muto, 2000); (Muramatsu et al. (1999) JBC 274: 18740-76; Muramatsu et al. (2000) Cell 102:553-64; Revy et al. (2000) Cell 102:565- 76). AID contains a ZDD (Zinc-dependent deaminase domain) and has 34% amino acid identity to APOBEC-1 (Table 4, Figure 5 and 6). Its location on human chromosome 12pl3 suggests it may be related to APOBEC-1 by a gene duplication event (Lau, 1994; Muto, 2000). This chromosomal region has been implicated in the autosomal recessive form of Hyper-IgM syndrome (HIGM2) (Revy, 2000). Most patients with this disorder have homozygous point mutations or deletions in three of the five coding exons, leading to missense or nonsense mutations (Revy, P., 2000) Cell. 102:565-75). Significantly, some patients had missense mutations for key amino acids within AED's ZDD (Revy, 2000; Minegishi, 2000).
37. AED homologous knockout mice demonstrated that AED expression was the rate limiting step for class switch recombination (CSR) and required for an appropriate level of somatic hypermutation SHM (Muramatsu, 2000). The expression of AED controls antibody diversity through multiple gene reanangements involving mutation of DNA sequence and recombination. The initial expression of antibodies requires immunoglobulin (Ig) gene reanangement that is AED-independent (Muramatsu, M., et al., (2000) Cell 102:553-63). This occurs in immature B lymphocytes developing in fetal liver or adult bone manow and requires DNA double strand breaks at the Ig heavy chain locus whose ends are rejoined by non-homologous end joining. The reananged immunoglobulin V (variable), D (diversity) and J (joining) gene segments encode a variable region that is expressed initially with the Attorney Docket Number 21108.0035P1
mu (μ) constant region (Cμ) to form a primary antibody repertoire composed of IgM antibodies. In humans and many mammals, AED-dependent gene alterations occur in B lymphocytes that are growing in germinal centers of secondary lymphoid organs following antigen activation. This involves multiple mutations of the variable region through Somatic Hypermutation (SHM) as well as removing the Cμ and replacing it with one of several other constant regions (Ca, Cd, Ce or Cg) through a recombination process known as Class Switch Recombination, CSR. In sheep, rabbits and chickens, pre-immune Ig gene diversification is mediated by an AED- dependent process known as gene conversion (GC) in which stretches of nucleotide sequences from one of several pseudogene V elements are recombined into the VDJ exon to generate diversity (Fugmann, S.D. et al., (2002) Science 295:1244-5.; Honjo, T., et al., (2002) Annu Rev Immunol. 20:165-96.).
38. Overexpression of AED in mouse fibroblasts and Ramos B cells induced CSR on an Ig reporter gene and stimulated the rate of SHM respectively (Muramatsu, M. et al. (2000) Cell. 102:553-63; Okazaki, LM. et al. (2002) Nature. 416:340-45). Given AED's similarity to APOBEC-1, these genomic alterations have been proposed to be due to AED-dependent niRNA editing (Muramatsu, 2000). Editing could promote CSH and SHM through the expression of a novel protein or by reducing the expression/function of an inhibitory protein through alternative exon splicing or codon sense changes.
39. AED cannot substitute for APOBEC-1 in the editing of apoB mRNA (Muramatsu, 1999) and, although this negative result may have been expected (given that most editing enzymes have substrate specificity (Grosjean and Benne (1998)), it did suggest that AED may have another activity. A competing hypothesis for AED's role in CSR and SHM is that it deaminates deoxycytidine in DNA (Rada, C. et al.
(2002) Proc. Natl. Acad. Sci USA. 99:7003-7008; Petersen-Mahrt, S.K., et al., (2002) Nature. 418:99-104). The mutations observed in SHM (and those that arise proximal to the junctions of CSR) are C-T transitions (Yoshikawa, K., et al., (2002) Science, 296:2033-2036). Like APOBEC-1, AJD has cytidine and deoxycytidine deaminase activity (Muramatsu, 1999) and its ZDD is homologous to that of E. coli deoxycytidine deaminase (Figure 5). Attorney Docket Number 21108.0035P1
40. AID overexpression in NTH 3T3 fibroblasts resulted in the deamination of deoxycytidine in DNA encoding a green fluorescent protein (GFP) (Yoshikawa, 2002) and also in antibiotic resistance and metabolic genes when AED expression in bacteria was placed under selection for a mutator' phenotype (Harris, 2002). A variety of mutations were observed on GFP DNA including deletions and duplications; however, a preference for transitions at G/C base pairs clustered within regions predicted to have DNA secondary structure was observed. Similar mutations were observed in the bacteria overexpressing AED and their frequency was markedly enhanced when evaluated in an ung-1 background (lacking functional uracil-DNA glycosylase, an enzyme involved in repairing C to T mutations). (Harris, 2002).
These findings together with the observation that the mutation frequency of the GFP gene was 4.5 x 10"4/bρ per cell generation, which was comparable to the 10"3 to 10"4 frequency observed on Ig genes in B cells, show that AED can act on DNA. The target hotspot for AED is characterized by the motif RGYW (R is A or G, Y is C or T and W is A or T) (Honjo et al. Annu Rev Immunol 20:165-96, 2002; Martin et al. Nat
Rev Immunol, 2(8):605-14, 2002).
41. No mutation hotspot was identified for APOBEC-1 and CEM15 although they have distinct substrate specificities (Harris et al. Mol Cell 10(5): 1247-53, 1996). Actively transcribed DNA was identified as the prefened AED substrate (Chaudhuri et al. Nature 422(6933):726-30, 2003), and specifically that dC is deaminated to dU in the sfrand of DNA that is displaced by transcription of RNA (the non-templating sfrand); conoborating other studies in which AID selectively deaminated dC in ssDNA or mutated dsDNA reporters within a nine base pair mismatch (the size of a transcription bubble) (Bransteitter et al. Proc Natl Acad Sci U S A 100(7) :4102-7, 2003; Ramiro et al. Nat Immunol, 2003). AED appears to act processively on DNA, binding initially to RGYW and mutating dC to dU and then modifying multiple dC residues from that point along the same sfrand of DNA. AID's ability to act on DNA would not negate the possibility that it also acts on RNA. Whether AID is involved in DNA and/or RNA modification, its function clearly results in the diversification of expressed genomic sequences.
Figure imgf000020_0001
Attorney Docket Number 21108.0035P1
3. APOBEC-2
42. Human APOBEC-2 (Genbank Accession # XM004087) is encoded on chromosome 6 and is expressed uniquely in cardiac and skeletal muscle (Liao et al. Biochem Biophys. Res. Commun. 260:398-404). It shares homology with APOBEC- I's catalytic domain, has a leucine/isoleucine-rich C-terminus and a tandem structural homology of the ZBD in its C-terminus. APOBEC-2 deaminated free nucleotides in vitro but did not have editing activity on apoB mRNA.
4. CEM15/APOBEC-3
43. Human phorbolin 1, phorbolin 1 -related protein, phorbolin-2 and -3 share characteristics with C to U editing enzymes. Several proteins with homology to
APOBEC-1 named Phorbolins 1, 2, 3, and Phorbolin-1 related protein were identified in skin from patients suffering from psoriasis and were shown to be induced (in the case of Phorbolins 1 and 2) in skin treated with phorbol 12-myristate-l -acetate (Muramatsu, M. et al. (1999) J Biol Chem. 274:18470-6). The genes for these proteins were subsequently renamed as members of the APOBEC-3 or ARCD family locus (Table 1) (Madsen, P. et al. (1999) J Invest Dermatol. 113:162-9). Bioinfoπnatic studies revealed the presence of two additional APOBEC-1 related proteins in the human genome. One is an expressed gene (XM_092919) located just 2 kb away from APOBEC-3 G, and is thus likely to be an eighth member of the family. The other is at position 12q23, and has similarity to APOBEC-3 G.
44. APOBEC-3 variants show homology to cytidine deaminases (Figure 6d). As anticipated from the SBSA, some of these proteins bind zinc and have RNA binding capacities similar to APOBEC-1 Jarmuz, A., et al, (2002) Genomics, 79:285-96).However, analysis of APOBEC-3A, -3B and -3G revealed them unable to edit apoB mRNA Jarmuz, A., et al, (2002) Genomics, 79:285-96); Muramatsu, M. et al. (1999) J Biol Chem. 274:18470-6). It has been shown that the frequency of deleterious mutations in HEV and impaired infectivity conelated with the expression of CEM15 (APOBEC-3G) (Sheehy et al, 2002; Mariani et al, 2003; Mangeat et al, 2003; Hants et al, 2003; Lecossier et al, 2003. HEV expressing functional Vif (viral infectivity factor) protein was able to overcome the effects of CEM15 due to the ability of Vif to bind and target fit or ubiquitinate and distract in the proteasome (Mariani et al., Cell 114:21-31, 2003; Stopal et al. Mol. Cell 12:591-601, 2003; Yu et Attorney Docket Number 21108.0035P1
al. Nat Struct Mol. Biol 11 :435-42, 2004). In confrast, it is unlikely that APOBEC-3D and 3E functions as an APOBEC-1 like editase because it is missing fundamental sequence elements that are required for mRNA editing by both APOBEC-1 and CDDl (Anant, S. et al. (2001) Am J Physiol Cell Physiol. 281.C1904-16; Dance et al 2001) and experimental evidence shows it has impaired ability to coordinate Zn2+ and deaminate cytidine Jarmuz, A., et al, (2002) Genomics, 79:285-96). APOBEC-3E appears to be a pseudogene (Jarmuz, A., et al, (2002) Genomics, 79:285-96), yet the EST database shows that APOBEC-3D and APOBEC-3E are alternatively spliced to fonn a single CD-PCD-CD-PCD encoding transcript. Additionally, it has been shown that rat APOBEC- 1 , mouse APOBEC-3 , and human APOBEC-3B, are able to inhibit HEV infectivity even in the presence of Vif. Like APOBEC-3G, human APOBEC-3F preferentially restrict vif-deficient virus. The mutation spectra and expression profile of APOBEC-3F indicate that this enzyme, together with APOBEC- 3G, accounts for the G to A hypermutation of pro viruses described in HEV-infected individuals (Bishop et al., Cun. Bio. 14:1392-1396, 2004). In accordance with this, it has also been shown that APOBEC-3F blocks HEV-l and is suppressed by both the HEV-l and HEV-2 Vif proteins (Zheng et al, J Virol 78(11): 6073-6076, 2004; Wiegand et al, EMBO 23:2451-58, 2004). The limited tissue expression, and association with pre-cancerous and cancerous cells (Table 1), and in the case of APOBEC-3G, antagonism of the HEV viral protein Vif shows specific roles for the APOBEC-3 family in growth cell cycle regulation and antiviral confrol.
45. APOBEC-3G (CEM15) has also been shown to interfere with other retroelements, including but not limited to hepatitis B virus (HBV) and murine leukemia virus (MLV). The methods and compositions described herein are useful with any of these viruses (Bishop et al., Cun. Bio. 14:1392-1396, 2004; Machida et al, PNAS 101(12):4262-67, 2004; Turelli et al., Science, 303:1829, 2004).
46. Table 1 shows APOBEC-1 and related proteins have been described previously (Anant, S., et al., Am J Physiol Cell Physiol. 281:C1904-16.; Dance, G.S., et al, (2001) Nucleic Acids Res. 29:1772-80.; Jarmuz, A., et al., (2002) Genomics. 79:285-96) and extended through amino acid similarity searches with the (1) hidden Markov modeling software SAM trained with CDDl, APOBEC-1, APOBEC-2, AED and Phorbolin 1, (2) PHI-BLAST, using the target patterns H(V/A)-E-X-X-F-X19- Attorney Docket Number 21108.0035P1
(JVV)-(T/V)-(W/C)-X-X-S-W-(S/T)-P-C-X-X-C (SEQ LD NO: 60) and (H/Q-X-E-X- X-F-X(19j30)-P-C-X(2> )-C (SEQ ED NO: 61) (Figure 6a, where X = any ammo acid) The gene name and its chromosomal location are indicated and the Accession number of the encoded protein listed. Equivalent/former names are derived from GenBank 5 (Anant, S., et al., (1998) Biol Chem. 379: 1075-81.; Sheehy, A.M., et al., (2002)
Nature 418:646-650.). The major tissues of expression are listed. More extensive listings, especially for neoplastic tissues, can be found in the LocusLink pages of Genbank for the individual ARPs which can be accessed from the Unigene Cluster entries. Other human APOBEC-1 Related Proteins (hsARP) (HsARP-6, HsARP-7, 10 HsARP-8, HsARP-10 and HsARP-11) only EST data exists as evidence of a final protein product.
Figure imgf000023_0001
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Al,θl-l: -2|6pαi NP_t0o7SQ CΛtuwo Ciii'tlttic & skijtt!||>.l lϊisis-le lϊs.ARP-l US 227457
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ΛID 12|>I3 NPJ3S57I -- lϊ lyiiiplω-j'l-s 1 ΛRP-2 lls.149342
APOΠE -3A 22HI11 NI"_ώ 63745 PhOiliΛliu-I π»3I94f) kc3iι(itω ifcs I I'sARP-3 U5..34SBSΛ
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PhflilH>liιι-l-i-l;ιteιHU6JQ-4) Cdluis (_[)ecιllelul)61l)S4
P θ<lι«Iιιι-2 JQ3UE74) .IOIΛP BEC- I3)
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ARC 0-3
AIH]BI '-3C/22i)'l3.l CAB-J5-t7l Phøiiiolin-I (API 65520) 5pi(S^ιιlt'5!v*'iι-iirI.'ilιyπιu≤. HsARP-5 Ui.fiSSS
I
N) NJ Λl'C)ϋl.<'-3D.'224l3.t BI-S417U hej((l ι£ eck caacβiS- HsARP-6 I ll:_tl' uπl^l APOBHr-3E.'22.il31 PSEUDOΠENE ΛRCD-6 AI'C')BEC-3Dϊ'3ii/22i|13.l NMJ-I529S Uh-tlui 11.ARP-7 APOBEC-3F,a2iil3 I BO 758984 ARCD-5 11 L Mβiu)c |g$ II.ARP-S
(ϊz l ui j- AP0BEC-3 !22(|13.1 NP 06S59-I PUoilidHn-likft-iuotein s leeiΛesiiβΛtattt/ilij'imi- IlsARP-9 Hs-250619 MDSIM9(AΛU242oS| PBf.s/t<ιlQ:jή,luιi!μc *I liBy 1 IsCHM 15 μimw'iiiiii-i eκ8~ϊ'pl;ια>ϊiϊl»;prύ$ϊϊιia .
22ιU3.l XP 032919 UsARP-19 XP J92919
12q23 XP It 5! 70 HsARP- 11
Mϋiiϊc MιnAPOBEC-l6F2 NPJΪ2436 Ssnall Ihϋ.atlsfc'tivβi'Siil-fcrt MmCDAR-1 Mm.3333
B lym hooyii-ykklliay MπιΛPOBEC-2/17 NP 033824 ci liuo & Λcliiml muscle Mm ARP- 1 Mm 27822 luai .'si ii
.MniAlDΛSiπ NP 033775 B IympI.i>--yr~S mΛR'P-2 Mm.3239S ran 5/15 Nl? 084531 XI? I22S5S πillinmϋijy tømoiir MnιARP-3 MmS 7<>
TABLE 1
Attorney Docket Number 21108.0035P1
47. Human HEV-l virus contains a 10-kb single-stranded, positive-sense RNA genome that encodes three major classes of gene products that include: (i) stractural proteins such as Gag, Pol and Env; (ii) essential trans-acting proteins (Tat, Rev); and
5 (iii) "auxiliary" proteins that are not required for efficient virus replication in at least some cell culture systems (Vpr, Vif, Vpu, Nef). Among these proteins, Vif is required for efficient viras replication in vivo, as well as in certain host cell types in vitro (Fisher et al. Science 237(4817):888-93, 1987; Strebel et al. Nature 328(6132):728-30, 1987) because of its ability to overcome the action of a cellular 10 antiviral system (Madani et al. J Virol 72(12):10251-5, 1998; Simon et al. Nat Med 4(12): 1397-400, 1998).
48. The in vitro replicative phenotype of vif-άeleted molecular clones of HEV-l is strikingly different in vz/-permissive cells (e.g. 293T, SUPT1 and CEM-SS T cell lines), as compared to vz -non-permissive cells (e.g. primary T cells,
15 macrophages, or CEM, H9 and HUT78 T cell lines). In the former cells, vif-deleted HEV-l clones replicate with an efficiency that is essentially identical to that of wild- type viras, whereas in the latter cells, replication of vz -negative HEV-l mutants is anested due to a failure to accumulate reverse transcripts and inability to generate infectious proviral integrants in the host cell (Sova et al. J Virol 67(10):6322-6, 1993;
20 von Schwedler et al. J Virol 67(8):4945-55, 1993; Simon et al J Virol 70(8):5297-
305, 1996; Courcoul et al. J Virol 69(4):2068-74, 1995). These defects are due to the expression of the host protein CEM15 (Sheehy, A.M., et al., (2002) Nature. 418:646- 650) in non-permissive cells for vz minus viruses. CEM15 antiviral activity is derived from effects on viral RNA or reverse transcripts (Sheehy, A.M., et al., (2002)
25 Nature. 418:646-650). CEM15 deaminates dC to dU as the first sfrand of DNA is being made by reverse transcriptase or soon after its completion, and this results in dG to dA changes at the conesponding positions during second strand DNA synthesis (Harris et al. Cell 113:803-809, 2003).
49. Primary sequence alignments (Figure 7) and the structural constraints
30 relating CDAs to APOBEC- 1 indicate that CEM15 evolved from an APOBEC- 1 -like precursor by gene duplication (Wedeldnd et al. Trends Genet 19(4): p. 207-16, 2003). The resulting CEM15 structure exhibits two active sites per polypeptide chain with Attorney Docket Number 21108.0035P1
the topology CDl-PCDl-connector-CD2-PCD2 (Figure 8). Knowledge of the structural homology among CD As and ARPs is sufficient to understand how features of CEMl 5 contribute to its anti-viral activity.
50. The premise of molecular modeling is that primary sequence analysis alone is insufficient to evaluate effectively the anti-viral activity of CEMl 5. The use of comparative modeling of CEMl 5 is based on three known CDA crystal structures (Betts et al. J Mol Biol 235(2):635-56, 1994; Johansson et al. Biochemistry 41(8): p. 2563-70, 2000) and knowledge gained from similar work with APOBEC-1. CEMl 5 modeling has been accomplished by aligning its amino acid sequence onto a composite three-dimensional template derived by superposition (Winn et al. J Synchrotron Radiat, (2003) 10(Pt l):23-5; Kabsch et al. Acta. Crystallogr., (1976) A32:922-923; Potterton et al. Acta Crystallogr D Biol Crystallogr, (2002) 58(Pt 11): p. 1955-7) of known crystal stractures, representing dimeric and teframeric quaternary folds of known CD As. The CEMl 5 sequence was modeled manually onto three dimensional template using the computer graphics package O (Jones et al. Acta Crystallogr A, (1991) 47 ( Pt 2): 110-9), thereby preserving the core ZDD fold; gaps and insertions were localized to loops and modeled according to one of the three known stractures, or by use of main-chain conformational libraries available in O; similarly amino acid side-chains rotamers were modeled using rotamer libraries (Jones et al. Acta Crystallogr A, (1991) 47 ( Pt 2):110- 9). Subsequently, a comparative model was created by use of the program 'Modeller' (Sali et al. Proteins, (1995) 23(3):318-26) and subsequently checked by Verify3D (Bowie et al. Science, (1991) 253(5016):164-70; Eisenberg et al. Methods Enzymol, (1997) 277:396-404). The model was energy minimized using simulated annealing and molecular dynamics methods including the CHARM2 energy parameters. No restraints were placed on secondary elements, except those derived from the triple CDA model alignment. The resulting model (Figure 9) demonstrates that the 384 amino acid sequence of CEMl 5 can be accommodated by a 222 CDA quaternary fold (analogous to the E. coli CDA or APOBEC-1 with 2 x 236 amino acids). Albeit CEM15 adopts a CD1-PCD1-CD2-PCD2 tertiary stracture with pseudo-222 symmetry (Figure 9) on a single polypeptide chain. The resulting CEMl 5 model provides a rational basis for the design of four classes of mutants: (id) active site zinc (Figure 6b) ligand changes " His65Ala (257), Cys97Ala (288), and CyslOOAla (291), (CD2 residues are noted Attorney Docket Number 21108.0035P1
parenthetically) and (ib) active site proton shuttle Glu57Gln (259). Notably, comparable type ( ) mutations in other CD As abolish activity (Carlow et al. Biochemistry, (1995) 34(13):4220-4; Navaratnam et al. J Mol Biol, (1998) 275(4):695-714; Kuyper et al. J. Crystal Growth, (1996) 168:135-169); (ii) substitution of the active site linker with a comparably sized linker sequence from E. coli. This change abolishes ACF-dependent mRNA editing activity by APOBEC- 1 in HepG2 cells. The linkers in the first and second active sites of CEMl 5 are conserved amongst ARPs. However, a 3 amino acid insert exists prior to the first linker in CEM15. The CEM15 model predicts mutation of the sequence of either linker would ablate activity whereas point modification of non conserved residues within insert should not; (iii) mutation of surface residues, e.g. F164 (F350) in the PCD(s) is predicted to disrupt auxiliary factor binding (but not mononucleoside deaminase activity), equivalent to the inactivating F156L mutation in APOBEC-1 (Navaratnam et al. J Mol Biol, (1998) 275(4):695-714). None of these mutations is expected to significantly disrupt the CEMl 5 polypeptide fold, but rather, will help localize regions of the structure necessary for anti- viral activity.
51. The number of possible CEMl 5 quaternary structures is limited (Figure 10); in fact evidence for a dimeric structure has been cited as 'unpublished' (Jarmuz et al. Genomics, (2002) 79(3):285-96). Therefore, a fourth class of mutants (truncations) are recognized that can be used to evaluate the requirement of single or dual CD domains for CEM15 activity. Possible dimeric CEM15 structures (Figure 10) predict mutually exclusive intermolecular contacts. The salient feature of the interaction depicted in Figure 10c, is that each CD pairs with itself, and similarly for each PCD. In contrast, every domain in Figure lOd falls in a unique environment (i.e. no CD or PCD pairs with itself). Therefore, to evaluate the need for either single or dual catalytic domain requirements for the anti-viral effect, trancations are expressed. For example, if the dual CD-PCD domain structure were required to ablate viral infectivity, truncation products of the form CD1-PCD1 or CD2-PCD2 would preclude folding of structures depicted in 10a, 10b and lOd, whereas model 10c could fold, leaving open the possibility that either CD1-PCD1 or CD2-PCD2 is sufficient to suppress viral infectivity. Therefore, anti- HEV-l therapeutics can be designed that disrupt Vif suppression of catalytic activity at either a single CD or both CD1 and CD2 simultaneously. The results of such mutations provide feedback, allowing a more rigorous refinement of the model by use of Modeller Attorney Docket Number 21108.0035P1
(Sali et al. Proteins, (1995) 23(3):318-26). Vif is known to have binding affinity for both viral RNA genomes and a variety of viral and cellular proteins (Simon et al. (1996) J. Virol. 70 (8):5297-5305; Khan et al. (2001) J. Virol. 75(16):7252-7265; Henzler et al. (2001) J. Gen Virol. 82: p. 561-573). Vif also can forms homodimers and homoteframers through its proline rich domain (Yang et al. (2002) J. Biol Chem. 278(8):6596-6602). The infectivity assay in the context of Vif minus pseudotyped viruses and 293 T cells either lacking or expressing CEMl 5 is found in Example 1. An assay was developed using VSV G-protein pseudotyped lentiviral particles that confirmed the inhibitory effect of CEMl 5 on the infectivity of vif+ and vif- HEV-l particles and is amenable to the rapid demarcation of the regions of HEV-l DNA (or RNA) that is the target for CEMl 5 catalytic activity.
52. Also, Vif interacts with CEM15 and induces its poly-ubiquitination and degradation through the proteosome, thereby reducing the abundance of CEMl 5 and promoting viral infectivity. It has been discovered that Vif homodimers were required for Vif s interaction with CEMl 5 (Yang et al. J Biol Chem. 278(8): 6596-602 (2003) and US Patent 6,653,443, herein incorporated by reference in their entirety).
53. It has been shown that a linker exists between catalytic domains of CEMl 5. Specifically, human CEMl 5 contains the amino acid residue "Asp" in the linker between the catalytic domains (Mariani et al. 2003; Bogerd et al. Proc. Natl Acad. Sci. 101 :3770-4, 2004; Zhang 2004). A negative to positive charge can be created if Asp or other negatively charged residues are replaced with a positively charged amino acid like lysine, arginine, or histidine, thereby abolishing the binding site of Vif on CEMl 5. Peptide or small molecule inhibitors that block or compete with binding of Vif to CEM15 can inhibit Vif s ability to block CEM15 activity. The CEM15 binding site of Vif can be similarly targeted, thereby achieving the same goal. Peptides or small molecules that bind the CEM15 binding site of Vif can similarly suppress Vif s effect on CEMl 5. Thus the Vif antagonists and methods for screening the same can be agents that block the CEMl 5 linker or the CEMl 5 binding site.
54. Agents that prevent Vif mediated polyubiquitination of CEMl 5 are also desired. Thus, Vif antagonists include agents that block the Vif-mediated polyubiquitination of CEM15. Vif interaction with CEM15 mediates CEM15's interaction with the polyubiquitination machinery, thereby leading to CEMl 5 Attorney Docket Number 21108.0035P1
conjugation with polyubiquitin (Yu et al Science 2003). This causes CEMl 5 to be shipped to the proteasome and degraded. By blocking polyubiquitination, CEMl 5 remains intact and can degrade the retrovirus. Peptides conesponding to the CEMl 5 sequence that contain the site of ubiquitination can act as mimetics to block 5 ubiquitination of CEM15. Such peptides can be delivered into cells via protein transduction using the aforementioned TAT sequence. Alternatively, Vif must interact with the ubiquitination machinery (Cul5-SCF complex; Yu et al. Science 2003, hereby incorporated by reference in its entirety) and peptide sequences of proteins in this complex can bind to Vif and thereby mimic and block the ability of Vif to target the 10 ubiquitination complex's binding to CEMl 5. These ubiquitin machinery mimetic peptides can be delivered into cells using the aforementioned protein transduction sequence of TAT.
55. Disclosed herein are polypeptides comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, or more contiguous amino acid residues of a
15 ubiquitination protein, wherein the polypeptide binds Vif and blocks ubiquitination of CEM15. Also disclosed is a polypeptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, or more contiguous amino acid residues of CEM15 wherien the polypeptide binds a ubiquitination protein and blocks Vif-mediated ubiquitination of CEMl 5. Also disclosed is a method of blocking the Vif-mediated ubiquitination of
20 CEMl 5 comprising contacting the CEMl 5 with the polypeptides disclosed herein.
56. Also disclosed is a polypeptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more or more contiguous amino acid residues of a CEM15 binding domain on Vif, wherein the polypeptide blocks CEMl 5-Vif interaction, as well as a method of blocking CEMl 5- Vif interaction comprising contacting Vif or CEM15
25 with the polypeptide disclosed above.
57. CEMl 5 contains a Gag binding domain. This binding domain allows for the CEMl 5 to be packaged into the virus. Vif, however, can block packaging from occurring. Thus, peptide mimetics resemblying the protein sequence of CEMl 5 that binds to Gag and the the CEMl 5 protein sequence that binds to Vif can interact with
30 Gag and Vif respectively and thereby block Gag and Vif from binding to CEM15. These peptide memetics enable CEMl 5 to enter the viral particle during its assembly
Figure imgf000029_0001
Attorney Docket Number 21108.0035P1
and prevent the distinction of CEMl 5, thereby ensuring ample CEMl 5 to be assembled with virions, respectively.
58. Disclosed is a polypeptide comprising 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous amino acid residues of a Gag protein,
5 wherein the polypeptide binds CEMl 5 and promotes CEMl 5 binding to viral RNA. Also disclosed is a method of promoting CEMl 5 binding to viral RNA comprising contacting CEMl 5 with the polypeptide disclosed herein.
59. Reverse transcription-dependent mutational activity of CEMl 5 on HEV-l ssDNA is not the only means by which CEMl 5 can reduce viral infectivity.
10 Mutations in one or both of the zinc- dependent cytidine deaminase domains did not ablate CEMl 5 's antiviral activity. Moreover, blockage of reverse transcriptase (RT) processivity by CEMl 5 binding to the viral RNA templates has been indicated as being an additional antiviral mechanism. In support of multiple mechanisms, transient expression of CEMl 5 reduced the level of pseudotyped HEV-l particles 15 generated from producer cells that were co- transfected with replication-defective proviral DNA constructs and helper plasmids.
60. Stably expressed CEMl 5 significantly reduced the level of pseudotyped HEV-l particles lacking Vif. The reduced viral particle production is the result of a selective suppression of viral RNA leading to reduction in essential HEV-l 20 proteins. These effects were not observed when Vif was expressed due to the marked reduction of CEMl 5. Although CEMl 5 was required to deplete viral particle production its deaminase function was not necessary. The data indicate an antiviral mechanism in producer cells which is potentially significant late during the viral life cycle that involves directly or indirectly the RNA binding ability of CEMl 5 and 25 does not require virion incorporation of CEMl 5 deaminase activity during viral replication. Thus, agents that enhance CEMl 5 selective binding to viral RNA, leading to viral RNA distraction result in a reduction in viral particle production and a reduced viral burden for the subject. Peptides conesponding to the portion of Gag protein sequence that binds to CEMl 5 can provide specificity to CEMl 5 for viral 30 RNA binding by CEMl 5. TAT transduction of these peptide mimetics activates CEMl 5 antiviral activity within cells. ,
Attorney Docket Number 21108.0035P1
61. The present invention may be understood more readily by reference to the following detailed description of prefened embodiments of the invention and the Examples included therein and to the Figures and their previous and following description. 5 62. Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods, specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for 10 the purpose of describing particular embodiments only and is not intended to be limiting.
B. Definitions
63. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.
15 Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such caniers, and the like.
64. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value.
20 Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is
25 also herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "10" is
30 disclosed the "less than or equal to 10"as well as "greater than or equal to 10" is also disclosed. Attorney Docket Number 21108.0035P 1
65. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
66. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
67. The terms "higher," "increases," "elevates," "enhances," or "elevation" refer to increases above basal levels, e.g., as compared to a confrol. The tenns "low," "lower," "reduces," "suppresses" or "reduction" refer to decreases below basal levels, e.g., as compared to a control. For example, basal levels are normal in vivo levels prior to, or in the absence of, addition of an agent such as a Vif antagonist or another molecule or ligand.
68. The term "test compound" is defined as any compound to be tested for its ability to bind to a Vif molecule, a deoxycytidine deaminase molecule, or a cytidine deaminase molecule. Examples of test compounds include, but are not limited to, small molecules such as K+, Ca2+, Mg2+ Fe2+ or Fe3+, as well as the anions SO4 2", H2PO " (or H3PO4) and NO ". Also, "test compounds" include drugs, molecules, and compounds that come from combinatorial libraries where thousands of such ligands are screened by drag class.
69. By "subject" is meant an individual. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. The term "subject" can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).
70. The terms "control levels" or "control cells" are defined as the standard by which a change is measured, for example, the confrols are not subjected to the experiment, but are instead subjected to a defined set of parameters, or the controls are based on pre- or post-treatment levels.
71. By "contacting" is meant an instance of exposure of at least one substance to another substance. For example, contacting can include contacting a substance, such as a cell, or cell to a test compound described herein. A cell can be contacted with the test compound, for example, by adding the protein or small molecule to the culture medimn (by continuous infusion, by bolus delivery, or by changing the medium to a medium that contains the agent) or by adding the agent to the extracellular fluid in vivo (by local
Figure imgf000032_0001
■
Attorney Docket Number 21108.0035P1
delivery, systemic delivery, intravenous injection, bolus delivery, or continuous infusion). The duration of contact with a cell or group of cells is determined by the time the test compound is present at physiologically effective levels or at presumed physiologically effective levels in the medium or extracellular fluid bathing the cell. In 5 the present invention, for example, a virally infected cell (e.g., a, HEV infected cell) or a cell at risk for viral infection (e.g., before, at about the same time, or shortly after HEV infection of the cell) is contacted with a test compound.
72. "Treatment" or "treating" means to administer a composition to a subject or a system with an undesired condition or at risk for the condition. The condition can be
10 any pathogenic disease, autoimmune disease, cancer or inflammatory condition. The effect of the administration of the composition to the subject can have the effect of but is not limited to reducing the symptoms of the condition, a reduction in the severity of the condition, or the complete ablation of the condition.
73. By "effective amount" is meant a therapeutic amount needed to achieve the 15 desired result or results, e.g., editing nucleic acids, interrupting CEMl 5-vif binding, reducing viral infectivity, inducing class switch recombination, somatic hypermutation, enhancing or blunting physiological functions, altering the qualitative or quantitative nature of the proteins expressed by cell or tissues, and eliminating or reducing disease causing molecules and/or the mRNA or DNA that encodes them, etc.
20 74. Herein, "inhibition" or "suppression" means to reduce activity as compared to a control (e.g., activity in the absence of such inhibition). It is understood that inhibition or suppression can mean a slight reduction in activity to the complete ablation of all activity. An "inhibitor" or "suppressor" can be anything that reduces the targeted activity. For example, suppression of CEMl 5-Vif binding by a disclosed
25 composition can be determined by assaying the amount of CEMl 5-Vif binding in the presence of the composition to the amount of CEMl 5-Vif binding in the absence of the composition and by decrease and increase (respectively) in viral infectivity. In this example, if the amount of CEMl 5- Vif binding is reduced in the presence of the composition as compared to the amount of CEMl 5-Vif binding in the absence of the
30 composition, the composition can be said to suppress the CEMl 5-Vif binding.
75. Many methods disclosed herein refer to "systems." It is understood that systems can be, for example, cells, columns, or batch processing containers (e.g., Attorney Docket Number 21108.0035P1
culture plates). A system is a set of components, any set of components that allows for the steps of the method to performed. Typically a system will comprise one or more components, such as a protein(s) or reagent(s). One type of system disclosed would be a cell that comprises both Vif and a test compound, for example. Another type of 5 system would be one that comprises a cell and an infective unit (e.g., an HEV unit). A third type of system might be a chromatography column that has CEMl 5, AED, or other deaminase or putative deaminase, bound to the column.
76. By "virally infected mammalian cell system" or "virally infected" is meant an in vitro or in vivo system infected by a virus. Such a system can include mammalian
10 cellular components; mammalian cells, tissues, or organs; and whole animal systems. By "HEV infectivity" or "viral infectivity" is meant the capacity of an in vitro or in vivo system to become infected by an virus (e.g., an HEV viras).
77. By "Vif antagonist" is meant any molecule or composition that counteracts, reduces, suppresses, inhibits, blocks, or hinders the activity of a Vif molecule or a
15 fragment thereof. This includes Vif dimerization antagonists, which reduce, suppress, inhibit, block, or hinder the dimerization of Vif. Any time a "Vif antagonist" is mentioned, this includes Vif dimerization antagonists. Also included are agents that block Vif binding to the CEMl 5, agents that block Vif-mediated polyubiquitination of CEMl 5, and the like.
20 78. By "cytidine deaminase activator" is meant any molecule or composition that enhances or increases the activity of a cytidine deaminase molecule or a fragment thereof. By cytidine deaminase activator is also meant deoxycytidine deaminase activator, ARP activator, or any related molecule.
79. By "deoxycytidine deaminase activator" is meant any molecule or
25 composition that enhances or increases the activity of a deoxycytidine deaminase molecule or a fragment thereof.
80. By "ARP activator" is meant any molecule or composition that enhances or increases the activity of an APOBEC-1 Related Protein molecule or a fragment thereof.
81. A "cytidine deaminase-positive cell" means any cell that expresses one ore 30 more cytidine deaminases or deoxycytidine deaminases. Such express can be naturally occurring or the cell can include an exogenous nucleic acid that encodes one ore more selected deaminases. Attorney Docket Number 21108.0035P 1
C. Screening methods
82. Disclosed herein are methods of screening for Vif antagonists, deoxycytidine deaminase activators, or cytidine deaminase activators. The method of screening for Vif antagonists comprises contacting a Vif molecule with a test compound; detecting binding between the Vif molecule and the test compound or detecting other desired interactions (such as CEM15-Vif binding or binding of Vif with proteins of the polyubiquitin machinery or block Gag interaction with CEM15); and screening the test compound that binds the Vif molecule or display another interaction for suppression of viral infectivity. Suppression of viral infectivity by the test compound indicates the test compound is a Vif antagonist. For the identification of Vif antagonists, it is not necessary to know whether Vif interacts with CEMl 5 or other viral or cellular proteins nor is it necessary to know the region(s) of Vif that is required to inhibit CEMl 5 activity.
83. Also provided is a method of screening for a Vif antagonist, comprising contacting a CEMl 5 molecule with a test compound; detecting binding between the
CEMl 5 molecule and the test compound or detecting other desired interactions (such as CEMl 5-Vif binding or binding of Vif with proteins of the polyubiquitin machinery or block Gag interaction with CEMl 5); and screening the test compound that binds the CEMl 5 molecule for its ability to block binding of Vif with the CEMl 5 or to suppress viral activity. An agent that blocks binding of Vif to CEM15 or displays other desired interactions is a Vif antagonist, which can be further tested for its ability to suppress viral infectivity.
84. As discussed above, "suppression" means to reduce activity as compared to a control (e.g., activity in the absence of such inhibition or suppression). It is understood that inhibition or suppression can mean a slight reduction in activity to the complete ablation of all activity. An "inhibitor" or "suppressor" can be anything that reduces activity. For example, suppression of CEMl 5- Vif binding by a disclosed composition can be determined by assaying the amount of CEMl 5-Vif binding in the presence of the composition to the amount of CEMl 5-Vif binding in the absence of the composition. In this example, if the amount of CEMl 5-Vif binding is reduced in the presence of the composition as compared to the amount of CEMl 5- Vif binding in the Attorney Docket Number 21108.0035P1
absence of the composition, the composition can be said to suppress the CEMl 5-Vif binding.
85. As disclosed in Example 4, an infectivity assay was carried out in the context of Vif minus pseudotyped virases and 293 T cells either lacking or expressing CEM15. The assay confirmed the inhibitory effect of CEM15 on the infectivity of vif+ and vif- HIV-1 particles. The results (Figure 12) indicate that the expression of CEMl 5 in 293T cells resulted in at least a 100-fold decrease in Vif- viral infectivity compared to particles generated in parental 293T cells. The low level of GFP expression from vif, CEMl 5+ particles is indistinguishable from background fluorescence in confrol cells.
86. This assay can be extended to include Vif + proviral DNA confrols and the use of deaminase inactivated CEMl 5 mutants in stable 293T cell lines. The assay is also amenable to the use of several existing HEV-l proviral isotyped vectors that are deleted for different regions and different amounts of the HEV-l genome, as well as to other retroviruses. Deleted genes can be provided in trans by co-transfection of suitable expression plasmids. A comprehensive examination of viral proteins and host tRNALys3 derived from Vif- virions revealed no significant biochemical or priming defects (Gaddis et al. J. Virol 77(10):5810-5820, 2003.) Dissection of such modifications can be performed in pseudotype viral assays in which key infectivity factors can be rapidly identified and assayed.
87. The screening assay described herein is useful for detecting Vif antagonists, deoxycytidine deaminase activators, or cytidine deaminase activators. These can block, prevent, or inhibit dimerization of Vif, block the Vif binding site for CEMl 5 or change the charge of CEMl 5 or compete with the CEM15/Vif binding sites to block or inhibit binding, block polyubiquitination, enhance CEMl 5 binding to viral RNA, or block Gag interaction with CEMl 5.
88. In one example, each cytidine deaminase activator, deoxycytidine deaminase activator, ARP activator, and Vif antagonist test compound can be tested by treating one or more of the cell types expressing a cytidine deaminase or deoxycytidine deaminase, or ARP, with each test compound and by infecting them with HIV-1 pseudotyped viras (or another refrovirus, or HCV or HBV, for example) containing GFP as described above. Within 48 hours post infection, cell culture Attorney Docket Number 21108.0035P 1
supernatants containing viral particles can be added to HeLa cells to test their infectivity, as evidenced by the appearance of green fluorescent cells in FACS analysis as described above. Reduction or elimination of green fluorescent cells relative to that observed in infections from producer cells that were not treated with cytidine deaminase activators or Vif antagonists are scored as a positive identification of cytidine deaminase activators, deoxycytidine deaminase activators, or Vif antagonist test compounds.
89. Vif antagonists, deoxycytidine deaminase activators, or cytidine deaminase activators enable the normal cellular amounts of CEMl 5 to mutate HEV-l, HCV, HB V, MLV, or any other retrovirus, to the extent that the virus cannot reproduce itself and therefore cannot elicit a productive infection. Vif antagonists enable CEMl 5 to mutate viral sequence at the level of first sfrand DNA synthesis and the resultant dC to dU change is templated during second strand DNA synthesis as dG to dA changes. The frequency of these changes is significantly greater than the mutation rate of reverse transcriptase and consequently the mutations in the retro viral genome affect numerous coding sequences at numerous positions, thereby rendering the viras nonfunctional (incapable of producing infectious virions).
90. The screening methods disclosed herein can be used with a high throughput screening assay, for example. The high throughput assay system can comprise an immobilized anay of test compounds. Alternatively, the Vif molecule or the cytidine deaminase molecule can be immobilized. There are multiple high throughput screening assay techniques that are well known in the art (for example, but not limited to, those described in Abriola et al., J Biomol. Screen 4:121-127, 1999; Blevitt et al., J Biomol. Screen 4:87-91, 2000; Hariharan et al., J Biomol. Screen 4:187-192, 1999; Fox et al., J Biomol. Screen 4:183-186, 1999; Burbaum and Sigal, Cun: Opin. Chem. Biol. 1:72-78, 1997; Jayasena, Clin. Chem. 45:1628-1650, 1999; and Famulok and Mayer, Curr. Top. Microbiol Immunol 243:123-136, 1999).
91. The Vif molecule, deoxycytidine deaminase activator or cytidine deaminase activator can be linked to a reporter, such as luciferase, GFP, RFP, or FITC, for example. Glow luminescence assays have been readily adopted into high throughput screening facilities because of their intrinsically high sensitivities and long-lived signals. The signals for chemiluminescence, bioluminescence, and colorimetric systems Attorney Docket Number 2110S.0035P1
such as luciferase and beta-galactosidase reporter genes or for alkaline phosphatase conjugates are often stable for several hours.
92. Several commercial luminescence and fluorescence detectors are available that can simultaneously inject liquid into single or multiple wells such as the WALLAC 5 VICTOR2 (single well), MICROBETA RTM JET (six wells), or AURORA VEPR (eight wells). Typically, these instruments require 12 to 96 minutes to read a 96-well plate in flash luminescence or fluorescence mode (1 mm/well). An alternative method is to inject the test compound/ Vif molecule/cytidine deaminase/deoxycytidine deaminase molecule into all sample wells at the same time and measure the
10 luminescence in the whole plate by imaging with a CCD camera, similar to the way that calcium responses are read by calcium-sensitive fluorescent dyes in the FLEPR or FLEPR-384 instruments. Other luminescence or fluorescence imaging systems include LEADSEEKER from AMERSHAM, the WALLAC VIEWLUX TM ultraHTS microplate imager, and the MOLECULAR DEVICES CLIPR imager.
15 93. PE BIOSYSTEMS TROPIX produces a CCD-based luminometer, the
NORTHSTAR TM HTS Workstation. This instrument is able to rapidly dispense liquid into 96-well or 384-well microtiter plates by an external 8 or 16-head dispenser and then can quickly transfer the plate to a CCD camera that images the whole plate. The total time for dispensing liquid into a plate and transferring it into the reader is about 10
20 seconds.
94. The Vif molecule and the reporter can also form a chimera. Purified recombinant Vif (e.g., HA/6His or Vif-CMPK-HA 6His, where CMPK is chicken muscle pyruvate kinase) conjugated with fluorescein isothiocyanate (FITC) or a fusion protein of Vif and GFP (see diagram below) can be used in high throughput screening
25 assays.
Figure imgf000037_0001
Attorney Docket Number 21108.0035P1
Figure imgf000038_0001
95. The Vif molecule can be represented by SEQ ID NO: 7, and the HA domain of the molecule can be represented by SEQ ED NO: 46. The Vif- HA/6-His molecule can be represented by SEQ ED NO: 54 as follows:
MENRWQVMEVWQVDI^MRIKTWKSLVKHHMYISKKAKEWVYRHHYESTHPR ISSEVH1PLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNTQVDP DLADKLIHLHYFDCFSDSAIRHAILGHRVRPKCEYQAGHNKVGSLQYLAL TALITPKKIKPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGHGYPYDVPD YAGHHHHHH
96. Designates a TEV protease cleavage site (or other appropriate protease cleavage site) where a proteolytic cleavage can be performed on recombinant Vif- CMPK so that Vif may be purified free of CMPK prior to its conjugation to FITC. Vif with or without CMPK may be produced depending on which protein produces the highest yield of soluble protein. A similar strategy can be used for Vif-GST, in which GST is glutathione-S-fransferase fused to the Vif N-terminus. Vif can be freed from the GST affinity tag by cleavage with PreScission™ protease, and is then suitable for fluorescein labeling. Regions 6His and HA are not drawn to scale. GFP can also be used in conjunction with the Vif molecule. Vif-GFP would not require a protease cleavage site due to its fluorescence; hence GFP- Vif would not require FITC conjugation. For cytidine deaminase or deoxycytidine deaminase activator or ARP activator HTS screening, Vif has been substituted with CEMl 5 in all of the constructs listed above. 97. The Vif-TEV-CMPK-HA 6-His molecule can be represented by SEQ ID
NO: 58 as follows:
MENRWQVMlVWQVDRMRlKTWKSLVi i MYISKKAKEWVYRHHYESTHPR ISSEVHEPLGDAKLVITTYWGLHTGEREWHLGQGVSEEWRKKRYNTQVDP DLADKLfflLIiYFDCFSDSAIRHAILGHRVRPKCEYQAGHNKVGSLQYLAL TALITPKKJT PPLPSVRKLTEDRWJ>JXPQKTKGHRGSHTMNGHGENLYFQG
MSKΉHDAGTAFIQTQQLHAAMADTFLEHMCRLDEDSEPTLARNTGΠCTI GPASRSVDKLKEMEKSGMNVARLNFSHGTHEYHEGTIKNVREATESFASD PITYRPVAEALDTKGPEERTGLEKGSGTAEVELKKGAALKVTLDNAFMEN CDENVLWVDYKNLEKVEDVGSKΓYVDDGLISLLVKEKGKDFVMTEVENGG MLGSKKGVNLPGAAVDLPAVSEKDIQDLKFGVEQNVDMVFASFLRKAADV HAVRKVLGEKGKHE SKMNHEGVRRFDELMEASDGEMVARGDLGIEI Attorney Docket Number 21108.0035P1
PAEKVFLAQKjMMGRCNJAAGl^πCATQMLESMlXE-PRPTj AEGSDVANA VLDGADCIMLSGETAKGDYPLEAVRMQHAIAREAEAAMFHRQQFEEILRH
SVHHREPADAMAAGAVEASFKCLAAALEVMTESGRSAHLVSRYRPRAPΠ AVTR )QTARQAHLYRGVFPVLCKQPAHDAWAEDVDLRVNLGMNVGKARG 5 FFKTGDLVIVLTGWL^GSGYTNTMRVVPWGYPYDVPDYAFFIHHHHHH
98. The Vif-TEV-EGFP-HA/6-His molecule can be represented by SEQ ED
NO: 56 as follows:
MENRWQVMIVWQVDRMRIKTWKSLVKHHMYISKKAKEWVYRHHYESTHPR ISSEVHEPLGDAKLVITTYWGLHTGEREWHLGQGVSIEWRKKRYNTQVDP 10 DLADKLIHLHYFDCFSDSAIRHAILGHRVRPKCEYQAGHNKVGSLQYLAL TALITPKKTKPPLPSVRKLTEDRWJNKPQKTKGHRGSHTMNGHGENLYFQG
MVSKGEELFTGVVPΓLVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKXPWWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIF FKDDGNYKTRAEVKFEGDTLVNRFFILKGLDFKEDGNILGHKLEYNYNSHN 15 VY1MADKQKNGIKVNFKIRHNIEDGS VQLADHYQQNTPIGDGP VLLPDNH YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKYPYDVPDYAHH
HHHH
99. In general, compounds that modulate the activity of Vif, deoxycytidine
20 deaminases, ARPs, or cytidine deaminases can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually
25 any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis
30 (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds (e.g., but not limited to, antibodies, peptides, and aptamers). Synthetic compound libraries are commercially available, e.g., from Brandon Associates (Menimack, NH) and Aldrich Chemical (Milwaukee, WI).
35 Disclosed is a method of screening for cytidine deaminase activators, comprising: contacting a cytidine deaminase molecule with a test compound; detecting binding between the cytidine deaminase molecule and the test compound; and screening the test Attorney Docket Number 21108.0035P1
compound that binds the cytidine deaminase molecule to identify a selected cytidine deaminase function, the presence of the selected function indicating a cytidine deaminase activator.
100. The cytidine deaminase molecule can be CEM15. Therefore, the cytidine 5 deaminase activator can be a CEMl 5 activator. The selected CEMl 5 function can be an increase, decrease, or any modification in the activity of the CEMl 5 or modifications in CEMl 5 interaction with other proteins (such as Vif) that modulate CEMl 5 deaminase activity. For example, the activity of CEMl 5, such as deoxycytidine to deoxyuridine mutation in the first strand of cDNA, can be increased upon binding of a test 10 compound, thereby decreasing or suppressing viral infectivity. Alternatively, the activity of CEMl 5 can be decreased, wherein the test compound binds CEMl 5 and the cytidine to uridine editing of mRNA or deoxycytidine to deoxyuridine mutation of DNA is inhibited or suppressed. A decrease in CEMl 5 activity can decrease its cancer promoting activity, or reduce cancer phenotype, in vitro or in vivo. An example of a 15 decrease in cancer promoting activity in the presence of compomids that bind CEMl 5 is found in breast cancer.
101. The ability of a test compound to suppress viral infectivity can be measured by contacting the test compound with a cytidine deaminase molecule in the presence of Vif and a virus. As disclosed above, the assays disclosed herein are useful 20 for detecting Vif antagonists, deoxycytidine deaminase activators, or cytidine deaminase activators. These can block, prevent, or inhibit dimerization of Vif, block the Vif binding site for CEMl 5 or change the charge of CEMl 5 or compete with the CEMl 5/ Vif binding sites to block or inhibit binding, block polyubiquitination, enhance CEMl 5 binding to viral RNA, or block Gag interaction with CEMl 5. 25 102. The CEMl 5 function can be, but is not limited to, its cytidine to uridine editing of RNA, or its deoxycytidine to deoxyuridine mutation of DNA, or its suppression of viral activity, or its activity on cancerous or precancerous cells. An "increase in CEMl 5 activity" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold increase in the function of the 30 CEM15. A "decrease in CEM15 activity" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold decrease in the function of the CEM15. Attorney Docket Number 21108.0035P1
103. The cytidine deaminase molecule can also be APOBEC-1. Therefore, the cytidine deaminase activator is an APOBEC-1 activator. In one example, the activity of APOBEC-1 can be increased such that the levels of apoB48 are increased due to cytidine to uridine editing of apoB mRNA and the levels of apoBlOO are consequently decreased as compared to a control level. Increasing APOBEC-1 activity can reduce atherogenic risk by promoting the activity of TAT- APOBEC-1 or the activity of APOBEC-1 expression from a transgene. Alternatively, the activity of APOBEC-1 can be decreased by binding of APOBEC-1 and the test compound, wherein the cytidine to uridine editing of mRNA or deoxycytidine to deoxyuridine mutation of DNA is inhibited or suppressed. An example of the decrease in cancer promoting activity in the presence of compounds that bind CEMl 5 is found in colon or rectal cancers.
104. The APOBEC-1 function can be, but is not limited to, its cytidine to uridine editing of RNA, or its deoxycytidine to deoxyuridine mutation of DNA, or the increased levels of apoB48 or decreased levels of apoBlOO as compared to a control, or its activity on cancerous or precancerous cells. An "increased levels of apoB48" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold increase in the level of apoB48 as compared to a confrol. A "decreased level of apoBlOO" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold decrease in level of apoBlOO as compared to a control.
105. The cytidine deaminase molecule can also be AED. Therefore, the cytidine deaminase activator is an AED activator. In one example, the activity of AED can be increased such that the levels of cytidine to uridine editing or the levels of deoxycytidine to deoxyuridine mutation are increased and the subsequent and consequent class switch recombination and or somatic hypermutation within the immunoglobulin locus of genes within B lymphocytes is increased. Increasing AID activity can enhance the immune response in individuals that are immunocompromised or have become immunodepressed. Increasing A D activity (for example, the AID activity that promotes class switch recombination) can also enhance the growth and proliferation of B cell lymphomas that express or overexpress AED or mutant forms thereof but fail to undergo class switch recombination or somatic hypermutation. Alternatively, the activity of AED can be decreased such that the levels of cytidine to Attorney Docket Number 21108.0035P1
uridine RNA editing or deoxycytidine to deoxyuridine mutation are decreased (for example, the AED activity that promotes somatic hypermutation), thereby reducing cancer promoting activity or cancer phenotype. An example of the decrease in cancer promoting activity in the presence of compounds that bind AJJD is found in the freatment of B cell lymphomas that express or overexpress AED, thereby creating inappropriate AED edited mRNAs or AED mutated DNA sequences, or mutant forms thereof. These cells may or may not have undergone class switch recombination or somatic hypermutation.
106. The AED function can be, but is not limited to, its cytidine to uridine editing of RNA, or its deoxycytidine to deoxyuridine mutation of DNA, or the promotion of antibody diversity produced by lymphocytes as compared to antibody production by control lymphocytes, or its activity on cancerous or precancerous cells. "Promotion of antibody diversity" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold increase in diversity of antibodies as compared to confrol lymphocytes. A "decreased level of AED" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100- fold, or 1000-fold decrease in level of AED as compared to a control.
107. The cytidine deaminase molecule can also be another ARP listed in Table 1. Therefore, the cytidine deaminase activator is an ARP activator. In one example, the activity of ARP can be increased such that the levels of cytidine to uridine editing or the levels of deoxycytidine to deoxyuridine mutation are increased and the subsequent encoded macromolecule affected by RNA editing or DNA mutation and the physiological process dependent on that native sequence of the affected macromolecule is modulated. RNA editing and DNA mutations induced by ARPs can have health promoting activities when appropriate regulated or disease causing activities when dysregulated. Disclosed herein are molecules that can enhance ARP activity through either direct binding to ARPs or by binding to the macromolecules that interact with ARP as natural regulators of ARP activity.
108. The ARP function can be, but is not limited to, the cytidine to uridine editing of RNA, or the deoxycytidine to deoxyuridine mutation of DNA, or the promotion of health-promoting or disease-causing pathways. Attorney Docket Number 21108.0035P1
109. As disclosed above in reference to the Vif antagonist, the cytidine deaminase can also be linked to a reporter, such as luciferase, GFP, RFP, or FITC, for example. The cytidine deaminase or Vif and the reporter can also form a chimera, as disclosed above. As disclosed above, the cytidine deaminase molecule can be CEMl 5, AED, APOBEC-1, or any other ARP molecule. The sequences conesponding to CEM15, AID, and APOBEC-1 are SEQ ED NOS: 1, 3, and 5, respectively. The conesponding nucleic acid sequences are SEQ ED NOS: 2, 4, and 6, respectively.
110. The disclosed compositions (e.g., Vif, cytidine deaminase, or their variants or fragments thereof) can be used as discussed herein as either reagents in micro anays or as reagents to probe or analyze existing microanays. The compositions can also be used in any known method of screening assays, related to chip/micro anays. The compositions can also be used in any known way of using the computer readable embodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions. 111. The effectiveness of the Vif antagonists or the cytidine deaminase activator can be assessed by detecting deaminase activity. Thus, levels of edited viral RNA and/or mutated (edited) viral DNA, wherein elevated levels of edited viral RNA or mutated (edited) viral DNA indicate enhanced deaminase activity. Additionally, levels of cellular RNA and DNA deaminases comprising by detecting levels of edited cellular RNA and/or mutated (edited) cellular DNA.
112. Disclosed are methods of identifying an inhibitor of an interaction between the deaminase and the viral infectivity factor, Vif, comprising incubating a library of molecules with the deaminase to form a mixture, and identifying the molecules that disrupt the interaction between the deaminase and the viral infectivity factor. There are many ways of disrupting the interaction between the deaminase and Vif, or the CEMl 5 interaction with Gag, such as blocking, preventing, or inhibiting dimerization of Vif; blocking the Vif binding site for CEMl 5 such as changing the charge of CEM15 or competing with the CEM15/Vif binding sites to block or inhibit binding; blocking polyubiquitination; enhancing CEMl 5 binding to viral RNA, or blocking Gag interaction with CEMl 5.
113. . An isolating step can comprise incubating the mixture with molecule comprising Vif or a fragment or derivative thereof. 0
Attorney Docket Number 21108.0035P1
114. Disclosed are methods of identifying an inhibitor or suppressor of an interaction between a deaminase and a viral infectivity factor (e.g., CEMl 5 and Vif, respectively) comprising incubating a library of molecules with the viral infectivity factor to form a mixture, and identifying the molecules that disrupt the interaction between the deaminase and the viral infectivity factor. The interaction disrupted can comprise an interaction between the viral infectivity factor and an amino acid of deaminase. An isolation step can comprise incubating the mixture with a molecule comprising a cytidine deaminase or fragment or derivative thereof. D. Compositions 115. Disclosed are Vif antagonists identified by the screening methods. Also disclosed are cytidine deaminase activators identified by the screening methods. Also disclosed are deoxycytidine deaminase activators identified by the screening methods. Also disclosed are ARP activators identified by the screening methods. The agents can function by interacting with Vif (e.g., Vif antagonist) or interacting with deoxycytidine deaminase or cytidine deaminase (e.g., cytidine deaminase activator). The Vif antagonist can bind or otherwise interact indirectly with Vif, thereby inhibiting its interaction with CEMl 5. This can include, but is not limited to, blocking, preventing, or inhibiting dimerization of Vif; blocking the Vif binding site for CEMl 5; changing the charge of CEMl 5 or competing with the CEM15/Vif binding sites to block or inhibit binding; blocking polyubiquitination; enhancing CEMl 5 binding to viral RNA, or blocking Gag interaction with CEMl 5.
116. . The cytidine deaminase activator or deoxycytidine deaminase activator can bind, or otherwise interact, with a cytidine deaminase or deoxycytidine deaminase, thereby enhancing the normal activity of the cytidine deaminase or deoxycytidine deaminase. For example, a cytidine deaminase activator can interact with CEMl 5 and enhance the binding of CEMl 5 to a virus. Conversely, a cytidine deaminase activator can interact with the binding of Vif to a CEMl 5 molecule, thereby suppressing the activity of Vif, and indirectly enhancing CEMl 5 binding to HEV. 117. The Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators of the invention can be modified to enhance suppression of viral activity or to lower biotoxicity. Such modification can 05/023
Attorney Docket Number 21108.0035P1
further enhance desirable properties, such as: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. 118. For example, the Vif antagonist or cytidine deaminase molecule can be modified following Lipinski's Rule of Five. Lipinski's Rule of Five is particularly useful when the goals of compound design are (i) to have less than 5 hydrogen donors, (ii) less than 10 hydrogen bond acceptors, (iii) molecular weight of less than 500 Daltons and (iv) the log of the partition coefficient, P (where P = the concentration of the compound in water divided by the concentration of the compound in 1 octanol) is less than 5. The Lipinski Rule of Five is an example of compound modification, however, the invention is not limited to these parameters. '
119. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. Also disclosed are the compositions identified by the methods disclosed therein.
120. In some cases the compositions of the invention are chimeric proteins. By "chimeric protein" is meant any single polypeptide unit that comprises two distinct polypeptide domains joined by a peptide bond, optionally by means of an amino acid linker, or a non-peptide bond, wherein the two domains are not naturally occurring within the same polypeptide unit. Typically, such chimeric proteins are made by expression of a cDNA construct but could be made by protein synthesis methods known in the art. These chimeric proteins are useful in screening compounds, as well as with the compounds identified by the methods disclosed herein. 121. The compositions disclosed herein can also be fragments or derivatives of a naturally occuning deaminase or viral infectivity factor. A "fragment" is a polypeptide that is less than the full length of a particular protein or functional domain. By "derivative" or "variant" is meant a polypeptide having a particular sequence that differs at one or more positions from a reference sequence. The fragments or derivatives of a full length protein preferably retain at least one function of the full length protein. For example, a fragment or derivative of a deaminase includes a fragment of a deaminase or a derivative deaminase (e.g., APOBEC-1, AED, CEMl 5, or Attorney Docket Number 21108.0035P1
an activator of a deaminase) that retains at least one binding or deaminating function of the full length protein. By way of example, the fragment or derivative can include a Zinc-Dependent Cytidine Deaminase domain or can include 20, 30, 40, 50, 60, 70 80, 90% similarity with the full length deaminase. The fragment or derivative can include conservative or non-conservative amino acid substitutions. The fragment or derivative , can include a linker sequence joining a catalytic domain (CD) to a pseudo-catalytic domain (PCD) and can have the domain stracture CD-PCD-CD-PCD or any repeats thereof. The fragment or derivative can comprise a CD. Other fragments or derivatives are identified by structure-based sequence alignment (SBSA) as shown herein. See Figure 6b that reveals the consensus stractural domain attributes of APOBEC-1 and ARPs (Figure 6c). The fragment or derivative optionally can form a homodimer or a homotetramer. Also disclosed are chimeric proteins, wherein the deaminase domain is a fragment or derivative of CEMl 5 having deaminase function.
122. "Deaminases" include deoxycytidine deaminase, cytidine deaminase, adenosine deaminase, RNA deaminase, DNA deaminase, and other deaminases.
Optionally, the deaminase is APOBEC-1 (see international patent application designated PCT/US02/05824, which is incorporated herein by reference in its entirety for APOBEC-1, chimeric proteins related thereto, and uses thereof) (Gen Bank Accession # NPJ301635), REE (see U.S. Pat. No. 5,747,319, which is incorporated herein by reference in its entirety for REE and uses thereof), or REE-2 (see U.S. Pat. No. 5,804,185, which is incorporated herein by reference in its entirety for REE-2 and uses thereof). Deaminases as described herein can include the following structural features: three or more CDD-1 repeats, two or more functional CDD-1 repeats, one or more zinc binding domains (ZBDs), binding site(s) for mooring sequences, or binding sites for auxiliary RNA binding proteins. Deaminases optionally edit viral RNA, host cell mRNA, viral DNA, host cell DNA or any combination thereof. One deaminase described herein is CEMl 5. CEMl 5 is homologous to Phorbolin or APOBEC-3G (see, for example, Accession #NP_068594). The names CEMl 5 and APOBEC-3G can be used interchangeably. CEMl 5 reduces retro viral infectivity as an RNA or DNA editing enzyme.
123. By "deaminating function" is meant a deamination of a nucleotide (e.g., cytidine, deoxycytidine, adenosine, or deoxyadenosine). Deaminating function is
Figure imgf000047_0001
Attorney Docket Number 21108.0035P1
detected by measuring the amount of deaminated nucleotide, according to the methods taught herein, wherein such levels are above background levels (preferably at least 1.5 - 2.5 times the background levels of the assay.)
124. Optionally, the Vif fragment or derivative thereof has at least 20, 30, 40, 5 50, 60, 70, 80, or 90 % amino acid similarity with the Vif molecule of SEQ ID NO: 7.
Optionally, the APOBEC-1 fragment or derivative thereof has at least 20, 30, 40, 50, 60, 70, 80, or 90 % amino acid similarity with the APOBEC-1 molecule of SEQ ED NO: 5. Optionally, the AID fragment or derivative thereof has at least 20, 30, 40, 50, 60, 70, 80, or 90 % amino acid similarity with the AED molecule of SEQ ID NO: 3. 10 Optionally, the CEM15 fragment or derivative has at least 20, 30, 40, 50, 60, 70, 80, or 90 % amino acid similarity with the CEMl 5 molecule of SEQ ED NO: 1.
125. It is understood that, as discussed herein, the use of the terms "homology" and "identity" are used interchangeably with "similarity" with regard to amino acid or nucleic acid sequences. Homology is further used to refer to similarities
15 in secondary and tertiary structures. In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of similarity to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of genes and proteins herein disclosed
20 typically have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 91, 98, or 99 percent similarity to the stated sequence or the native sequence. For example, SEQ ED NO: 2 sets forth a particular nucleic acid sequence that encodes a CEMl 5, and SEQ ED NO: 1 sets forth particular sequences of the proteins encoded by those nucleic acids. Also, SEQ ED
25 NOS: 4, 6, and 8 sets forth particular nucleic acid sequences that encode an ALD, an APOBEC-1, and a Vif protein, respectively, and SEQ ED NOS: 3, 5, and 7 sets forth particular sequence of the proteins encoded by those nucleic acids. Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
30 91, 92, 93, 94, 95, 96, 97, 98, 99 percent similarity to the stated sequence. Those of skill in the art readily understand how to detennine the similarity of two proteins or Attorney Docket Number 21108.0035P1
nucleic acids, such as genes. For example, the similarity can be calculated after aligning the two sequences so that the similarity is at its highest level.
126. Another way of calculating similarity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the alignment algorithm of Needleman and Wunsch, J. Mol Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WE), or by inspection.
127. The same types of similarity can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.
128. For example, as used herein, a sequence recited as having a particular percent similarity to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent similarity to the second sequence as calculated by any of the other calculation methods. As another example, a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent similarity to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods. As yet another example, Attorney Docket Number 21108.0035P1
a first sequence has 80 percent similarity, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent similarity to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated similarity percentages). 5 129. Other structural similarities aside from sequence similarity are also disclosed. For example, homology, as noted by similar secondary and tertiary stracture, can be analyzed as taught herein. Homologous proteins may have minimal sequence similarity but have a homologous catalytic domain. Thus, deaminases as used herein maybe structurally similar based on the structure of the catalytic domain or other
10 domain but have lower than 70% sequence similarity.
130. Vif antagonists as well as cytidine deaminase activators, deoxycytidine deaminase activators, and ARP activators can be identified using variants and derivatives of cytidine deaminases, deoxycytidine deaminases, or Vif. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino
15 acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for
20 example, on the order of one to four residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein
25 sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a
30 known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about
Figure imgf000050_0001
Attorney Docket Number 21108.0035P1
from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary RNA stracture. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 2 and 3 and are refened to as conservative substitutions.
10 131. TABLE 2 : Amino Acid Abbreviations
Figure imgf000050_0002
TABLE 3: Amino Acid Substitutions
Original Residue Exemplary Conservative Substitutions
Ala; Ser
Arg; Lys; Gin
Asn; Gin; His
Asp; Glu
Cys; Ser Gin; Asn, Lys Attorney Docket Number 2110S.0035P1
Glu; Asp
Gly; Pro
His; Asn; Gin lie; Leu; Val
Leu; He; Val
Lys; Arg; Gin;
Met; Leu; He
Phe; Met; Leu; Tyr
Ser; Thr
Thr; Ser
Trp; Tyr Tyr; Trp; Phe Val; He; Leu
132. Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 3, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the stracture of
5 the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic
10 residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this
15 case, (e) by increasing the number of sites for sulfation and/or glycosylation.
133. For example, the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions
20 include combinations such as, for example, Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein. Attorney Docket Number 2110S.0035P1
134. Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
135. Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the conesponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of ' the C-terminal carboxyl.
136. The compositions disclosed herein can be used as targets in combinatorial chemistry protocols or other screening protocols to isolate molecules that possess desired functional properties related to inhibition of the CEMl 5-Vif, activation of cytidine deaminase or deoxycytidine deaminase, or antagonism of Vif activity. 137. Given the infomiation herein, molecules that function like the disclosed molecules can be identified and used as discussed herein. For example, the knowledge that CEMl 5 interacts with Vif indicates targets for identifying molecules that will affect retroviral infectivity. Disclosed are compositions and methods of making these compositions that bind Vif, such that CEMl 5 binding to Vif is competitively inhibited or suppressed. Also disclosed are compositions and methods of making these compositions that bind (or interact with) cytidine deaminase molecules, such as CEMl 5. Preferably, the molecules enhance or suppress a cytidine deaminase or deoxycytidine deaminase function. As discussed herein, this knowledge can be used along with, for example, combinatorial chemistry techniques, identify molecules that function as desired, by for example, inhibiting or suppressing CEMl 5 and Vif binding, or mimic other cytidine deaminases or deoxycytidine deaminases. Attorney Docket Number 21108.0035P1
138. The disclosed compositions, such as cytidine deaminases or deoxycytidine deaminases (e.g., CEM15, APOBEC-1, AED, and other ARPs) or Vif can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired
5 way or mimic their function. The nucleic acids, peptides, and related molecules disclosed herein can be used as targets for the combinatorial approaches.
139. It is understood that when using the disclosed compositions in combinatorial techniques or screening methods, molecules, such as macromolecular molecules, will be identified that have particular desired properties such as inhibition,
10 suppression, or stimulation or the target molecule's function. The molecules identified and isolated when using the disclosed compositions, such as, CEMl 5, AED, APOBEC- 1, ARPs, or Vif, are also disclosed. Thus, the products produced using the combinatorial or screening approaches that involve the disclosed compositions, such as, CEMl 5, AED, APOBEC-1, ARPs, or Vif are also disclosed. Such molecules include
15 Vif antagonists and cytidine deaminase activators.
140. Combinatorial chemistry includes but is not limited to all methods for isolating small molecules or macromolecules that are capable of binding either a small molecule or another macromolecule like Vif or cytidine deaminase (e.g., CEMl 5), typically in an iterative process. Proteins, oligonucleotides, and sugars are examples of
20 macromolecules. For example, oligonucleotide molecules with a given function, catalytic or ligand-binding, can be isolated from a complex mixture of random oligonucleotides in what has been refened to as "in vitro genetics" (Szostak, TLBS 19:89, 1992). One synthesizes a large pool of molecules bearing random and defined sequences and subjects that complex mixture, for example, approximately 1015
25 individual sequences in 100 μg of a 100 nucleotide RNA, to some selection and enrichment process. Through repeated cycles of affinity chromatography and PCR amplification of the molecules bound to the ligand on the column, Ellington and Szostak (1990) estimated that 1 in 1010 RNA molecules folded in such a way as to bind a small molecule dyes. DNA molecules with such ligand-binding behavior have been
30 isolated as well (Ellington and Szostak, 1992; Bock et al, 1992). Techniques aimed at similar goals exist for small organic molecules, proteins, antibodies and other macromolecules known to those of skill in the art. Screening sets of molecules for a Attorney Docket Number 21108.0035P1
desired activity whether based on small organic libraries, oligonucleotides, or antibodies is broadly refened to as combinatorial chemistry. Combinatorial techniques are particularly suited for defining binding interactions between molecules and for isolating molecules that have a specific binding activity, often called aptamers when the 5 macromolecules are nucleic acids.
141. There are a number of methods for isolating proteins that either have de novo activity or a modified activity. For example, phage display libraries have been used to isolate numerous peptides that interact with a specific target (United States Patent No. 6,031,071; 5,824,520; 5,596,079; and 5,565,332 which are herein
10 incorporated by reference in their entirety for their material related to phage display and methods relate to combinatorial chemistry).
142. A prefened method for isolating proteins that have a given function is described by Roberts and Szostak (Roberts R. W. and Szostak J.W. Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997). This combinatorial chemistry method couples the
15 functional power of proteins and the genetic power of nucleic acids. An RNA molecule is generated in which a puromycin molecule is covalently attached to the 3 '-end of the RNA molecule. An in vitro translation of this modified RNA molecule causes the conect protein, encoded by the RNA to be translated. In addition, because of the attachment of the puromycin, a peptdyl acceptor which cannot be extended, the growing
20 peptide chain is attached to the puromycin which is attached to the RNA. Thus, the protein molecule is attached to the genetic material that encodes it. Normal in vitro selection procedures can now be done to isolate functional peptides. Once the selection procedure for peptide function is complete traditional nucleic acid manipulation procedures are performed to amplify the nucleic acid that codes for the selected
25 functional peptides. After amplification of the genetic material, new RNA is transcribed with puromycin at the 3 '-end, new peptide is translated and another functional round of selection is perfonned. Thus, protein selection can be performed in an iterative manner just like nucleic acid selection techniques. The peptide which is translated is controlled by the sequence of the RNA attached to the puromycin. This
30 sequence can be anything from a random sequence engineered for optimum translation (i.e. no stop codons etc.) or it can be a degenerate sequence of a known RNA molecule to look for improved or altered function of a known peptide. The conditions for nucleic Attorney Docket Number 21108.0035P1
acid amplification and in vitro translation are well known to those of ordinary skill in the art and are preferably performed as in Roberts and Szostak (Roberts R. W. and Szostak J.W. Proc. Natl. Acad. Sci. USA, 94(23)12997-302 (1997)).
143. Another prefened method for combinatorial methods designed to isolate 5 peptides is described in Cohen et al. (Cohen B.A., et al, Proc. Natl. Acad. Sci. USA
95(24): 14272-7 (1998)). This method utilizes and modifies two-hybrid technology. Yeast two-hybrid systems are useful for the detection and analysis of protei protein interactions. The two-hybrid system, initially described in the yeast Saccharomyces cerevisiae, is a powerful molecular genetic technique for identifying new regulatory
10 molecules, specific to the protein of interest (Fields and Song, Nature 340:245-6 (1989)). Cohen et al. modified this technology so that novel interactions between synthetic or engineered peptide sequences could be identified which bind a molecule of choice. The benefit of this type of technology is that the selection is done in an intracellular environment. The method utilizes a library of peptide molecules that
15 attached to an acidic activation domain. A peptide of choice, for example a portion of Vif is attached to a DNA binding domain of a franscriptional activation protein, such as Gal 4. By performing the Two-hybrid technique on this type of system, molecules that bind the extracellular portion of Vif can be identified.
144. Using methodology well known to those of skill in the art, in
20 combination with various combinatorial libraries, one can isolate and characterize those small molecules or macromolecules, which bind to or interact with the desired target. The relative binding affinity of these compounds can be compared and optimum compounds identified using competitive binding studies, which are well known to those of skill in the art.
25 145. Techniques for making combinatorial libraries and screening combinatorial libraries to isolate molecules which bind a desired target are well known to those of skill in the art. Representative techniques and methods can be found in but are not limited to United States patents 5,084,824, 5,288,514, 5,449,754, 5,506,337, 5,539,083, 5,545,568, 5,556,762, 5,565,324, 5,565,332, 5,573,905, 5,618,825,
30 5,619,680, 5,627,210, 5,646,285, 5,663,046, 5,670,326, 5,677,195, 5,683,899, 5,688,696, 5,688,997, 5,698,685, 5,712,146, 5,721,099, 5,723,598, 5,741,713, 5,792,431, 5,807,683, 5,807,754, 5,821,130, 5,831,014, 5,834,195, 5,834,318, Attorney Docket Number 21108.0035P1
5,834,588, 5,840,500, 5,847,150, 5,856,107, 5,856,496, 5,859,190, 5,864,010, 5,874,443, 5,877,214, 5,880,972, 5,886,126, 5,886,127, 5,891,737, 5,916,899, 5,919,955, 5,925,527, 5,939,268, 5,942,387, 5,945,070, 5,948,696, 5,958,702, 5,958,792, 5,962,337, 5,965,719, 5,972,719, 5,976,894, 5,980,704, 5,985,356, 5 5,999,086, 6,001,579, 6,004,617, 6,008,321, 6,017,768, 6,025,371, 6,030,917, 6,040,193, 6,045,671, 6,045,755, 6,060,596, and 6,061,636.
146. Combinatorial libraries can be made from a wide anay of molecules using a number of different synthetic techniques. For example, libraries containing fused 2,4-pyrimidinediones (United States patent 6,025,371) dihydrobenzopyrans
10 (United States Patent 6,017,768and 5,821,130), amide alcohols (United States Patent 5,976,894), hydroxy-amino acid amides (United States Patent 5,972,719) carbohydrates (United States patent 5,965,719), l,4-benzodiazepin-2,5-diones (United States patent 5,962,337), cyclics (United States patent 5,958,792), biaryl amino acid amides (United States patent 5,948,696), thiophenes (United States patent 5,942,387), tricyclic
15 Tetrahydroquinolines (United States patent 5,925,527), benzofurans (United States patent 5,919,955), isoquinolines (United States patent 5,916,899), hydantoin and thiohydantoin (United States patent 5,859,190), indoles (United States patent 5,856,496), imidazol-pyrido-indole and imidazol-pyrido-benzothiophenes (United States patent 5,856,107) substituted 2-methylene-2, 3-dihydrothiazoles (United States
20 patent 5,847, 150), quinolines (United States patent 5,840,500), PNA (United States patent 5,831,014), containing tags (United States patent 5,721,099), polyketides (United States patent 5,712,146), morpholino-subunits (United States patent 5,698,685 and 5,506,337), sulfamides (United States patent 5,618,825), and benzodiazepines (United States patent 5,288,514).
25 147. As used herein combinatorial methods and libraries include traditional screening methods and libraries as well as methods and libraries used in interative processes.
148. The disclosed compositions (including the Vif antagonists, deoxycytidine deaminase activators, ARP activators, and the cytidine deaminase
30 ' activators) can be used as targets for any molecular modeling technique to identify either the structure of the disclosed compositions or to identify potential or actual molecules, such as small molecules, which interact in a desired way with the disclosed Attorney Docket Number 21108.0035P1
compositions. The compounds disclosed herein can be used as targets in any molecular modeling program or approach.
149. It is understood that when using the disclosed compositions in modeling techniques, molecules, such as macromolecular molecules, will be identified that have
5 particular desired properties such as inhibition, suppression, or stimulation or the target molecule's function.
150. One way to isolate molecules that bind a molecule of choice is tlirough rational design. This is achieved through structural information and computer modeling. Computer modeling technology allows visualization of the three-
10 dimensional atomic stracture of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target
15 molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule- compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design
20 program and the user.
151. Examples of molecular modeling systems are the CHARMm and QUANTA programs, Polygen Corporation, Waltham, MA. CHARMm performs the energy minimization and molecular dynamics fimctions. QUANTA performs the construction, graphic modeling and analysis of molecular stracture. QUANTA allows
25 interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
152. A number of articles review computer modeling of drugs interactive with specific proteins, such as Rotivinen, et al., 1988 Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57 (June 16, 1988); McKinaly and Rossmann, 1989
30 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122; Perry and Davies, OSAR: Ouantitative Stracture- Activity Relationships in Drag Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. Lond. 236, 125-140 and 141-162; and, with respect Attorney Docket Number 21108.0035P1
to a model enzyme for nucleic acid components, Askew, et al., 1989 J Am. Chem. Soc. Ill, 1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc., Pasadena, CA., Allelix, the, Mississauga, Ontario, Canada, and Hypercube, hie, Cambridge, Ontario. Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of molecules specifically interacting with specific regions of DNA or RNA, once that region is identified.
153. Although described above with reference to design and generation of compounds which can alter binding, one can also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which alter substrate binding or enzymatic activity.
154. Also described is a compound that is identified or designed as a result of any of the disclosed methods can be obtained (or synthesized) and tested for its biological activity, e.g., competitive inhibition or suppression of CEMl 5-Vif binding or inhibition or suppression of retro viral infectivity.
155. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular CEMl 5, Vif, AID, APOBEC, Vif antagonist, deoxycytidine deaminase activator, ARP activator or cytidine deaminase activator is disclosed and discussed and a number of modifications that can be made to a number of molecules are discussed, specifically contemplated is each and every combination and permutation of thereof. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional
Figure imgf000059_0001
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steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
156. Also contemplated are various molecules with different binding sites on the deoxycytidine deaminase or cytidine deaminase and/or the regulatory proteins thereof that interact with the deoxycytidine deaminase or cytidine deaminase activators, inhibitors, or antagonists, and enhance or inhibit activity thereof. E. Methods of using the compositions
157. Disclosed are methods of interrupting viral infectivity (e.g., refroviral infectivity like HEV infectivity) comprising contacting an infected cell or a cell prior to infection with a Vif antagonist, under conditions that allow delivery of the antagonist into the cell, wherein the antagonist binds with a viral infectivity factor (Vif) or CEMl 5 to interrupt viral infectivity. h terraption of viral infectivity may occur at different levels, including, for example, at the level of RNA on the incoming virus, on first or second strand cDNA, after dsDNA integration and/or on transcripts from the viral integrin.
158. By "interrupting viral infectivity" is meant stopping or reducing the production of infective viral genomes. HIV infectivity, for example, is known to depend on a variety of proteins leading to the synthesis of double stranded DNA from single sfranded HEV RNA genome and the integration of HEV DNA into the host cell's chromosomal DNA from where it is expressed to form viral genomes and viral proteins necessary for virion production. A Vif antagonist reduces the ability of virion Vif to inactivate cellular processes, thus allowing CEMl 5 to effectively mutate HEV or alters its replication and chromosomal integration by affecting the editing of a cellular mRNA encoding a protein that blocks the production of infectious HEV.
159. The Vif antagonists, deoxycytidine deaminase activators, and cytidine deaminase activators described herein can work in a multitude of ways to interrupt viral infectivity. For example, they can block, prevent, or inhibit dimerization of Vif; block the Vif binding site for CEMl 5 or change the charge of CEMl 5 or compete with the CEM15/Vif binding sites to block or inhibit binding; block polyubiquitination; enhance CEMl 5 binding to viral RNA; or or block Gag interaction with CEMl 5. 05 0
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160. The disclosed compositions can be delivered to the target cells in a variety of ways. For example, the compositions can be delivered through elecfroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
161. Thus, the compositions can comprise, for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.
162. Disclosed are methods of treating a subject with a viral infection (e.g., HEV infection) or at risk for an infection comprising administering to the subject an effective amount of the Vif antagonist. As used throughout, administration of an agent described herein can be combined with various others therapies. For example, a subject with HEV may be treated concomitantly with protease inhibitors and other agents.
163. Also disclosed are methods of treating a subject with a viral infection or at risk of an infection with the compounds as described above. The compound can be in water soluble form, and can be administered by the various routes described throughout. One example of administration is oral administration.
164. A cytidine deaminase activator is an agent that enhances the efficiency of editing. Additional genetic, pharmacologic, or metabolic agents or conditions also modulate the RNA or DNA editing or mutating function of the deaminase. Some of the conditions that modulate editing activity include: (i) changes in the diet, (ii) hormonal changes (e.g., levels of insulin or thyroid hormone), (iii) osmolarity (e.g., hyper or hypo osmolarity), (iv) ethanol, (v) inhibitors of RNA or protein synthesis and (vi) conditions Attorney Docket Number 21108.0035P1
that promote liver proliferation. Thus, the methods of the invention can comprise administering a cytidine activator to the subject and using other conditions that enhance the efficiency of mRNA editing function.
165. Also disclosed are methods of treating a condition, wherein the 5 condition is a cancer. The cancer can be selected from the group consisting of lymphomas (Hodgkins and non-Hodgkins), B cell lymphoma, T cell lymphoma, myeloid leukemia, leukemias, mycosis fungoides, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic
10 tumours, myelomas, AEDS-related lymphomas or sarcomas, metastic cancers, bladder cancer, brain cancer, nervous system cancer, squamous cell carcinoma of head and neck, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary
15 cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, hematopoietic cancers, testicular cancer, colo-rectal cancers, prostatic cancer, or pancreatic cancer.
166. Also disclosed are methods wherein the condition to be treated is an infectious disease (e.g., a viral disease). Also disclosed are methods, wherein the viral
20 infection can be selected from the list of virases consisting of Herpes simplex viras type-1, Herpes simplex virus type-2, Cytomegalovirus, Epstein-Ban viras, Varicella- zoster virus, Human herpesvirus 6, Human herpesvirus 7, Human herpesviras 8, Variola viras, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C viras, Hepatitis D viras, Hepatitis E viras, Rhinovirus, Coronavirus, Influenza virus
25 A, Influenza virus B, Measles viras, Polyomaviras, Human Papilomaviras, Respiratory syncytial viras, Adenovirus, Coxsackie virus, Dengue viras, Mumps viras, Polioviras, Rabies viras, Rous sarcoma viras, Yellow fever viras, Ebola virus, Marburg viras, Lassa fever viras, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever
30 virus, Rotavirus A, Rotaviras B, Rotavirus C, Sindbis virus, Simian Immunodeficiency ciras, Human T-cell Leukemia viras type-1, Hantavirus, Rubella virus, Simian wo 2005/023 '
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Immunodeficiency virus, Human Immunodeficiency virus type-1, Vaccinia virus, SARS virus, and Human Immunodeficiency viras type-2.
167. Also disclosed are methods of treating a bacterial infection. The bacterial infection can include M. tuberculosis, M. bovis, M. bovis strain BCG, BCG
5 subsfrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species, Yersinia pestis, Pasteur ella haemolytica, Pasteur ella multocida, other Pasteur ella species, Actinobacillus pleuropneumoniae,
10 Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetti, other Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichia coli, Vibrio cholerae, Campylobacter
15 species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.
168. Also disclosed are methods of treating a parasitic infection. The 20 parasitic infection can include Toxoplasma gondii, Plasmodiumfalciparum,
Plasmodium vivax, Plasmodium malariae, other Plasmodium species., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species., Schistosoma mansoni, other Schistosoma species., and Entamoeba histolytica.
169. Also disclosed are methods of treating a fungal infection. The fungal 25 infection can include Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicillium marnefft, and Alternaria alternata.
170. Vif antagonists, deoxycytidine deaminase activators, ARP activators, 30 and cytidine deaminase activators are of benefit to individuals who are infected as well as to those who have recently been infected or anticipate an exposure to the virus. As new virases are produced in individuals who are HEV positive, or positive 2005/023985 " .
Attorney Docket Number 21108.0035P1
for another retrovirus, Vif antagonist, deoxycytidine deaminase activator, ARP activator, or cytidine deaminase activator freatment will induce mutations as virus infects new cells. Many of the mutated viruses are destroyed by host cell DNA repair mechanism. Those mutated viras that integrate into chromosomal DNA are not able 5 to produce infectious viral particles. The overall effect is reduced viral shedding into body fluids and consequently a reduction in the probability that new contacts with infected individuals will be infectious. Therefore Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators reduce the production of infectious viruses in affected individuals thereby controlling the disease
10 at an early stage and reducing the probability of transmission. For individuals who have been recently exposed or anticipate an exposure (rape victims, a child born to an HJV positive mother, healthcare workers, emergency personnel, disaster management teams, tenorist response teams and paramedics,) Vif antagonists, deoxycytidine deaminase activators, ARP activators, or cytidine deaminase activators can prevent a
15 productive infection from taking place by allowing CEMl 5 to destroy retro viral genomes before they can be integrated, or rendering those that do integrate nonproductive during their replication.
171. With all of the methods described herein, the virus can be a retrovirus (e.g., HIV). The virus can be an RNA virus. The RNA virus can be selected from the
20 list of virases consisting of Vesicular stomatitis virus, Hepatitis A virus, Hepatitis C viras, Rhinoviras, Coronavirus, Influenza virus A, Influenza virus B, Measles viras, Respiratory syncytial viras, Adenovirus, Coxsackie viras, Dengue viras, Mumps viras, Polioviras, Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus,
25 St. Louis Encephalitis virus, Munay Valley fever virus, West Nile virus, Rift Valley fever viras, Rotaviras A, Rotaviras B, Rotaviras C, Sindbis virus, Hantaviras, and Rubella virus.
172. The ability to suppress viral infectivity can be measured by contacting the test compound with one or more cytidine deaminase-positive cells, in the presence
30 of Vif and a virus. Cytidine deaminase positive cells are cells that express a cytidine deaminase molecule or fragment thereof, such as CEMl 5, APOBEC-1, AID, or ARPs. Attorney Docket Number 21108.0035P1
173. Thus, the disclosed compositions can also be used diagnostic tools related to diseases that are susceptible to RNA or DNA editing, such as HEV, HCV, HBV, or MLV.
174. As described above, the compositions can also be administered in vivo in 5 a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material maybe administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The 10 carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
175. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, fransdermally,
15 extracorporeally, topically or the like, although topical infranasal administration or administration by inhalant is typically prefened. As used herein, "topical infranasal administration" means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. The latter
20 may be effective when a large number of animals is to be treated simultaneously.
Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age,
25 weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
30 176. Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid Attorney Docket Number 21108.0035P1
prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. 177. The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol, hnmunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin- coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in wliich the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concenfration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers 178. Delivery of the Vif antagonist, deoxycytidine deaminase activator, ARP activator, or cytidine deaminase activator compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier. Pharmaceutical carriers are Attorney Docket Number 21108.0035P 1
known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
179. Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. 180. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic freatment is desired, and on the area to be treated. Administration may be topically (including opthamalically, vaginally, rectally, infranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed compounds can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
181. Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous earners include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
182. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Attorney Docket Number 21108.0035P1
183. Compositions for oral adnrinistration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
184. Some of the compositions may potentially be administered as a
5 pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an 10 inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. b) Therapeutic Uses
185. The dosage ranges for the administration of the compositions are those
15 large enough to produce the desired effect in which the symptoms disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual
20 physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
186. Vif antagonists, deoxycytidine deaminase activators, ARP activators, or cytidine deaminase activators that do not have a specific pharmaceutical function, but which may be used for tracking changes within cellular chromosomes or for the
25 delivery of diagnostic tools for example can be delivered in ways similar to those described for the pharmaceutical products.
187. As described previously, molecules such as Vif antagonists, deoxycytidine deaminase activators, ARP activators, and cytidine deaminase activators can be administered together with other forms of therapy. For example, the molecules
30 can be administered with antibodies, antibiotics, or TAT peptides. TAT-fusion peptides are especially useful with the methods described herein, as they are rapidly internalized by lipid raft-dependent macropinocytosis and then able to escape. dTAT-HA2 is also Attorney Docket Number 21108.0035P1
useful with the methods disclosed herein, and is fransduciblβ, pH-sensitive, and fusogenic (Wadia et al., Nature Medicine, 10(3) :310-315, 2004). F. Methods of making the compositions
188. The compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
189. Also disclosed are methods of making a Vif antagonist, comprising identifying a Vif antagonist by the screening methods disclosed herein; and modifying the Vif antagonist to enhance suppression of viral infectivity. Methods of modifying the Vif antagonist are disclosed herein. The Vif antagonist can be modified by a number of means, as disclosed above, such as using Lipinski's Rule of Five. Such modifications can include amino acid modifications, thereby producing variants and derivatives that enhance suppression of viral activity. Also disclosed are Vif antagonists and cytidine deaminase activators made by the methods described herein. 190. Disclosed are methods of making a cytidine deaminase activator comprising identifying the cytidine deaminase activator; and modifying the cytidine deaminase activator to enhance the selected deaminase function of the modified cytidine deaminase activator as compared to the function of the unmodified cytidine deaminase activator. Methods of modifying the cytidine deaminase activator are disclosed herein, such as using Lipinski's Rule of Five. The cytidine deaminase activator can be modified by a number of means, as disclosed above. Such modifications can include amino acid modifications, thereby producing variants and derivatives that enhance suppression of viral activity. The same method can be used to make deoxycytidine deaminase activators and ARP activators. 191. "Suppression of viral activity" is defined as a 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold suppression of viral activity. Viral activity includes, but is not limited to, viral reproduction, viral shedding, or viral infectivity.
192. Also disclosed are methods of making a Vif antagonist, comprising identifying the Vif antagonist by the screening methods disclosed herein; and modifying the Vif antagonist to lower biotoxicity of the test compound.
Figure imgf000069_0001
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193. Also disclosed is a method of making a cytidine deaminase activator comprising identifying the cytidine deaminase activator; and modifying the cytidine deaminase activator to lower biotoxicity of the modified cytidine deaminase activator as compared to the biotoxicity of the unmodified cytidine deaminase activator. The same
5 method can be used to make deoxycytidine deaminase activators and ARP activators.
194. "Lower biotoxicity" is defined as a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 10-fold, 100-fold, or 1000-fold lowering of the biotoxicity of the test compound. Biotoxicity is defined as the toxicity of the compound to a cell or to a system, in vitro or in vivo.
10 195. Disclosed are methods of treating a subject comprising administering to the subject an inhibitor of viral infectivity (e.g., HEV infectivity), wherein the inhibitor reduces the interaction between a deaminase (e.g., CEMl 5) and a viral infectivity factor (Vif), and wherein the subject is in need of such freatment.
196. Disclosed are methods of manufacturing a composition for inhibiting the 15 interaction between a deaminase (e.g., CEMl 5) and a viral infectivity factor (Vif) comprising synthesizing the Vif antagonists as disclosed herein. Also disclosed are methods of manufacturing a composition for enhancing the activity of a deaminase such as CEM15, APOBEC-1, AED, or other ARPs. Also disclosed are methods that include mixing a pharmaceutical carrier with the Vif antagonists, deoxycytidine deaminase 20 activator, ARP activator, or the cytidine deaminase.
197. Disclosed are methods of making a composition capable of inhibiting infectivity (e.g., HEV infectivity) comprising admixing a compound with a pharmaceutically acceptable carrier, wherein the compound is identified by the methods described herein.
25 G. Chips and microarrays
198. Disclosed are chips comprising nucleic acids that encode Vif, cytidine deaminases, deoxycytidine deaminases, ARPs, or fragments or variants thereof or where at least one address is such a nucleic acid. Also disclosed are chips where at least one address is an amino acid sequence for Vif, deoxycytidine deaminases, ARPs,
30 cytidine deaminases, or fragments or variants thereof. W
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H. Examples
199. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds,
5 compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some enors and deviations should be accounted for. Unless indicated otherwise, parts are 10 parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. U.S. Provisional Application No. 60/401,293 and PCT/US02/05824, are incorporated herein by reference in their entireties for the examples, methods, and compounds therein.
1. Example 1 15 a) Methods for obtaining the CEM15 cDNA and for cloning it into two different systems:
200. Human CEM15 (NP-068594; also known as MDS019, AAH24268) was amplified from total cellular RNA of the NALM-6 cell line (human B cell precursor leukemia) by RT-PCR.
20 Oligo-dT primed first-strand cDNA was amplified using Expand HiFi Taq DNA polymerase (Roche) with the following primers; '5 'A' CACTTTAGGGAGGGCTGTCC (SEQ ID NO: 10) and '3 'A* CTGTGATCAGCTGGAGATGG (SEQ ED NO: 11). Thel366 bp product was reamplified with CEMl 5 specific PCR primers that included Ncoϊ and IioI restriction
25 sites on the 5' and 3' primer respectively; '5'B'
CTCCCATGGCAAAGCCTCACTTCAGAAACACAG (SEQ ID NO: 12) and '3'B' CTCCTCGAGGTTTTCCTGATTCTGGAGAATGGCCC (SEQ ED NO: 13). The 1154bp PCR product was digested with EcoRL to remove potentially co-amplified highly homologous APOBEC3B/Phorbolin 3 (Q9UH17) sequences and the NcolVXhoI
30 digested product subcloned into a modified pET28a (Novagen) plasmid such that a CEM15-thrombin-HA-6His fusion protein could be expressed. The full-length human CEMl 5 cDNA was subcloned by PCR into a mammalian expression vector (pcDNA3) -
Attorney Docket Number 21108.0035P1
such that it is expressed with an amino terminal haemagglutinin (HA) epitope. It was also subcloned into ρET28a (Novagen) to express a 6His-thrombin-CEM15 fusion protein.
201. The expression of the former clone in mammalian HepG2 cells (Human liver hepatoma line) demonstrate expression of full length protein (PAGE gel cell extracts were transfened to nitrocellulose and the presence of CEMl 5 was determined by reaction with anti-HA tag antibodies). This latter fusion was expressed to high levels in E. coli as a soluble protein and purified by nickel affinity chromatography (the expression and yield of CEMl 5 was determined by Coomassie blue stained PAGE gel and was approximately 700 μg per 50 mis of original E. coli culture). 2. Example 2 a) APOBEC-1 model.
202. The construction of the APOBEC-1 model is based upon the hypothesis that enzymes with a common, catalytic function (i.e. hydrolytic deamination of a nucleoside base) exhibit a common tliree-dimensional fold despite a low overall amino acid sequence identity (even at levels <30%). This level of homology is often cited as the lower limit upon which one can reliably model the fold of a given polypeptide sequence (Burley, S.K. (2000) Nature Struct. Biol. 7:932-934.). However, the stractures of molecules with similar biological functions are known to be highly conserved even at low levels of primary stracture homology (Chothia et al. Embo J. 5(4)823-6, 1986; Lesk et al. J Mol Biol, 136(3):225-70.) At present experimentally derived three-dimensional structures are available for three cytidine deaminases (CD As) whose role in pyrimidine metabolism has been firmly established. These enzymes encompass the dimeric CDA from E. coli (Betts L. et al., CW (1994) J Mol Biol. 235:635-56), the teframeric CDA from B. subtilis (Johansson E. et al. (2002). Biochem.41 :2563-70) and the teframeric CDA Cddl from S. cerevisiae [Xie et al, & Wedekind, manuscript in preparation]. The Cartesian coordinates for the former two models are available in the public Protein Data Bank (www.rcsb.org/pdb) as entries 1 AF2 and 1 JTK. Among the known CDA stractures however, only Cddl exhibits RNA editing activity (Dance, G.S.C. et al. (2001) Nuc. Acids Res. 29:1772-1780.) and therefore its coordinates have been critical in the assembly of a composite 3-D model for APOBEC-1 because it provides direct evidence that the fundamental CDA polypeptide fold is necessary and sufficient for RNA editing and can
Figure imgf000072_0001
05/023985
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function as a dC to dU DNA mutator as evidenced by the activity of APOBEC-1 and CEM15. Furthermore, the Cddl crystal structure is a critical component in the development of a working model for RNA editing by APOBEC-1 and provides a tool to understand and manipulate its related proteins (ARPs) including AID, and CEMl 5. b) Methods for the Construction of a Structure-Based
Sequence Alignment (SBSA) Leading to a New APOBEC-1
Three-dimensional Model.
(1) Expression and Purification Cddl was amplified by PCR from Baker's yeast. The product was cloned into a pET-28a vector (Novagen) containing N-tenninal 6xHis using Ndel and EcoRI restriction sites; constructs were verified by DNA sequencing. BL21 CodonPlus (Stratagene) cells transformed with vector were grown at 37°C to an OD600 of 0.7 and induced with 1 mM EPTG at 30°C for 3 hours. Bacterial pellets were resuspended in lysis buffer (50 mM Tris-Cl pH 8.0, 10 mM β-mercaptoethanol, 1 mg/ml lysozyme, lmM PMSF, 2 mM benzamidine and 5μg/ml each of aprotinin, leupeptin and pepstatin A), lysed, and nuclease digested (0.5% Triton X-100, 2 mM ATP, 10 mM MgSO4, 33 μg/ml each of DNasel and RNasel) at 4°C. The 6xHis tagged protein was purified in batch with NiNTA agarose (Qiagen) utilizing the following wash, elution, and dialysis scheme: wash 7, 10 mM Tris-Cl pH 8.0, 100 mM KCl, 20 mM imidazole, 10% glycerol; wash 2, same as wash 1 including 1 M KCl; wash 3, repeat wash 1; elution, 10 mM Tris-Cl pH 8.0, 0.5 M KCl, 0.4 M imidazole, 10% glycerol; dialysis against 2x 2 liters 10 mM Tris-Cl pH 8.0, 120 mM NaCl, 1 mM DTT). Removal of the 6xHis tag was achieved by digestion for 16 hours at 20°C with 10 U biotinylated thrombin (Pierce). Protein was dialyzed against 20 mM HEPPS pH 8.0, 0.25 M KCl, 5% glycerol, and 4 mM DTT and concentrated to 6 mg/ml as estimated by Bradford assays (BioRad) using an Ulfrafree-4 spin cartridge (Millipore). Protein was utilized immediately for crystallization.
(2) Crystallization
204. Crystals were grown at 20°C from well solutions of 16.5% (w/v) PEG monomethylether (MME) 5K, 450 mM NH4C1, 100 mM Na-succinate pH 5.5, 10 mM DTT and 1 mM NaN3 by use of the hanging drop vapor diffusion method. Four μl of well solution was added to an equal volume of protein. Crystals appeared in six days Attorney Docket Number 21108.0035P1
and reached a maximum size of 50 x 90 x 450 mm3 after 3-4 weeks. Single crystals were harvested with a nylon loop (Hampton Research), and cryo-protected through four serial transfers in 100 μl volumes of solutions containing 19% (w/v) PEG monomethylether 5000, 500 mM NH4CI, 100 mM Na-succinate pH 5.5, 1 mM DTT and either 5, 10, 15 or 17.5% (v/v) PEG 550 MME. Crystals were flash cooled by plunging into liquid nitrogen, and stored for X-ray data collection. In order to bind UMP, crystals were serially transfened in the presence of 10 mM UMP from pH 5.5 to 7.5 in 0.5 pH unit increments. Buffers of the appropriate pKa were chosen for each step. Crystals were subsequently cryo-adapted at elevated pH and flash frozen as described. (3) Structure Determination
205. Crystals of scCddl belong to space group C222j with unit cell dimensions a = 78.51 A, b = 86.32 A and c - 156.14 A. There is one 16.5 kDa tetramer (4x 145 amino acids) per asu. The stracture was solved by use of MAD phasing at the Zn(π) K-absorption edge with the peak energy at 1.2828 A, inflection = 1.28310 A and remote energy 1.25740 A. The positions of four zinc atoms were located by use of the program SOLVE v2.0, and phases were density modified by use of the program RESOLVE with 4-fold NCS averaging. The NCS averaged phases improved electron density maps significantly and allowed manual skeletonization by use of O. Additional NCS averaging with DM improved maps quality and allowed modeling of amino acids 4 to 136 in all four subunits. Upon addition of UMP, the C-terminal 6 amino acids are observed. The present structure has been refined by use of CNS using all data from 30 to 2.0 A resolution with a crystallographic Rfactor of 23.2% (Rfree = 26.2%). The model exhibits reasonable bond and angle deviations from ideal values (0.009A and 1.52o, respectively) as evaluated by PROCHECK. More than 89% of residues are in the allowed region of the Ramachandran Plot.
(4) Homology Modeling
206. The design of homology models for the ARP enzymes was based upon the observation that the enzyme Cddl from Saccharomyces cerevisiae is capable of acting on monomeric nucleoside substrates of pyrimidine metabolism, as well as larger RNA substrates such as reporter apoB mRNA expressed ectopically in yeast (Dance et al, 2001 Nucleic Acid Res. 29, 1772-1780). These results along with our X-ray crystallographic structure determination of yeast Cddl demonstrated that the
Figure imgf000074_0001
0
Attorney Docket Number 21108.0035P 1
fundamental CDA fold, typical of pyrimidine metabolism enzymes, are sufficient for catalyzing C to U editing of RNA or dC to dU mutations on DNA. As such, the three known crystal structure of cytidine deaminases were utilized to prepare a template for homology modeling of APOBEC-1, CEMl 5 and AID. The initial amino acid sequence alignment among enzymes of known structure with those of the unknown ARPs was prepared by use of the program ClustalX vl.8 (Thompson et al., 1997 Nucleic Acid Res. 24, 4876-4882). Sequences aligned included: #P19079 (B. subtilis), #NP_013346 (S. cerevisiae), #1065122 (E. coli), #4097988 (APOBEC-1 from H. sapiens), NP_065712(AED from H. sapiens) and #NP_068594 (APOBEC-3G from H. sapiens), which were retrieved from the NCBI (www.ncbi.nlm.mh.gov Pubmed). Subsequently, manual adjustments were made to the alignments of the ARP primary sequences according to sequence constraints derived from the triple three-dimensional structural superposition of the known cytidine deaminase coordinates of yeast (i.e. scCDDl), E. coli (PDB accession number 1 AF2) and B. subtilis PDB (PDB accession number 1 JTK) described by Betts et al. (1994, J. Mol. Biol 235, 635-56) and Johansson et al. (2002 Biochemistry 41, 2563-70) as implemented in the program LSQKAB (Kabsch 1976 Acta Crystallogr. A 32, 922-923). When optimized to account for the conserved three- dimensional fold, the alignments between the enzymes of pyrimidine metabolism and the ARPs revealed sequence identity ranging from ~7% to 26% in the respective catalytic and non-catalytic domains. Despite the modest sequence identity at the amino acid level, the actual three-dimensional structural homology of proteins with a common function often far exceeds the relatedness values predicted by simple amino acid sequence alignments (Chothia & Lesk, 1986 EMBO J. 5, 823-826). In order to rigorously model the respective ARP structures with the highest degree of empirically derived structural restraints, the method of comparative modeling was employed using "satisfaction of spatial restraints" as implemented in the program Modeller (Sali & Blundell 1993, J. Mol. Biol. 234, 779-815). Following model calculation, realistic model geometry is achieved through real-space optimization using enforced stereochemical refinement derived from application of the CHARM22 force field parameters (MacKerell et al., 1998 J. Phys. Chem. B. 102 3586-16). In all models, the Zn2+ ion was constrained in Modeller to be within 2.25 A distance of each the respective putative metal ligands: 2x cyteine-Sγ and lx histidine-Nδl. This constraint Attorney Docket Number 21108.0035P1
resulted in a satisfactory and realistic tetrahedral geometry consistent with the known CDA stractures, as well as the chemical requirements for base hydrolytic deamination. hi order to model the location of DNA or RNA substrate binding, the edited nucleotide was modeled according to constraints derived from the known locations of CDA inhibitors in the template X-ray crystal stractures: 1 JKT (tetrahydrouridine ) and 1AF2 (3,4 dihydrouridine). Due to the known substrates of AID and APOBEC-1, DNA and RNA sequences were modeled as single-stranded. Additionally, the restraint that nucleotide bases flanking the edited/mutated sites maintain modest base stacking was imposed by adding additional distance restraints in the model calculation. Each monomer of a respective ARP model was also restrained to be symmetric. This method of modeling far exceeds previous standards employed to model APOBEC-1 (Navaratnam, N. et al. (1998) JMB 275:695-714.). The surprising result of modeling is the existence of an extensive flexible linker that extends from residues 136 to 143 of human APOBEC-1 and residues 131-138 of human AID. (5) Mutagenesis and Construction of Chimeric Cddl
Enzymes 207. hi order to corroborate the comparative model of APOBEC-1, the Cddl was employed as a model compound to examine: (i) the feasibility of the predicted APOBEC-1 fold, and (ii) the role of key functional elements predicted to be in the active site linker or other active site locations necessary for catalysis. (Mutations can be divided into two classes: those that stabilize/destabilize the structure through insertions or changes of large stretches of amino acids; and those that effect function by modest changes to amino acids). A series of mutants were constructed in a manner analogous to the following method. In order to assess the importance of the predicted C-terminal "tail" of Cddl upon the ability to edit RNA, a 19 amino acid linker from E. coli was added after residue 142. Specifically, Cddl was PCR amplified using a 5' Cddl -specific primer and a 3' primer encoding the 19 amino acid E. coli "linker" extension and subcloned into the Ndel and EcoRI sites of pET28a (Novagen). In order to assess the importance of linker flexibility Glyl37 was converted to Ala using the QuikChange mutagenesis system (Stratagene) according to the manufacturer's protocols; other point mutations were constructed similarly. To assesses whether or not the CDA from E. coli (PDB #1 AF2) was competent to edit under conditions similar to ,
Attorney Docket Number 21108.0035P1
APOBEC-1 and Cddl in yeast (Dance et al, 2001 Nucleic Acid Res. 29, 1772-1780; Dance et al., 2000 Nucleic Acids Res. 28, 424-9), the E. coli CDA was PCR amplified from genomic DNA and subcloned for yeast expression as described below. In order to address the question of whether or not the proposed homology model for APOBEC-1 (above) was feasible in terms of the overall three-dimensional fold and catalytic activity, a series of Cddl cliimeras were assembled by fusing together two Cddl polypeptide chains joined by a linker. The 5' monomers containing the appropriate C- terminal APOBEC-1 or E. coli 19 amino acid linker were amplified and subcloned as described above. The amino terminally foreshortened C-terminal monomer (missing helix αl based upon homology modeling) was PCR amplified using the wild type or Glu63 to Ala Cddl template and ligated as an EcoRlVXhoI fragment to the appropriate 5' monomer in pET28a. The linking EcoRI site was mutagenized to restore the reading frame of the Cddl chimeras. All Cddl monomer and chimeric cDNAs were amplified using Cddl specific primers and subcloned via EcoRI and Xbal sites into a modified pYES2.0 vector to allow galactose regulated expression of an HA-epitope tagged protein in yeast for Western analysis. Cddl mutants and chimeric proteins were expressed and purified essentially as described above. The results of editing in the context of the yeast system established for APOBEC-1 and Cddl (Dance et al 2001 Nucleic Acid Res. 29, 1772-1780; Dance et al., 2000 Nucleic Acids Res. 28, 424-9) are summarized in Figure 13.
208. In the context of late log phase growth in yeast with galactose feeding, overexpressed Cddl is capable of C to U specific editing of reporter apoB mRNA at site C6666 at a level of 6.7%, which is ~10x times greater than the negative confrol (Figure 13, empty vector - compare lanes 1 and 2, above). In confrast, the CDA from E. coli (equivalent to PDB entry 1AF2) is incapable of editing on the reporter subsfrate (Figure 13, lane 3). Similarly, the active site mutants E61A and G137A abolish detectable Cddl activity (Figure 13, lanes 4 and 5). Likewise, the addition of the E. coli linker sequence (Figure 13, lane 6) impairs editing function as well. In a series of chimeric constracts in which the Cddl tetramer was converted into a molecular dimer, the chimeric molecule appears functional, as long as an amino acid linker of 7-8 amino acids is used to join the respective Cddl subunits (Figure 13, Right Panel lanes 1-4). However, when the longer E. coli linker is used to join Cddl monomers, there is no Attorney Docket Number 21108.0035P 1
detectable activity on the reporter substrate, although the chimeric protein is expressed (Figure 13, Western blot). Paradoxically, when conserved Gly residues of the APOBEC-1 linker (130 and 138) are mutated to Ala, the chimeric enzyme is still active (Figure 13, lanes 3 and 4 of right panel). This shows that these components are not an important part of the linker flexibility, or that the new chimera adopts a different fold in this region compared to that of the pyrimidine metabolism enzymes. Indeed, the ARP models suggest a re-structuring of the active site linker that makes the entire region spanning from 130 to 142 (human APOBEC-1 numbering) flexible in a manner that moves to accommodate large polymeric substrates such as RNA or DNA (See AID active site model bound to DNA 9-mer BELOW). Additional evidence of the importance of the linker sequence comes from mutagenesis on rat APOBEC-1 (highly homologous to human). When the 8 amino acid linker sequence of rat APOBEC-1 is replaced with the first 8 amino acids of the E. coli linker, the APOBEC-1 construct is unable to edit reporter apoB mRNA in the human hepatoma cell line HepG2 (Navaratnam, N. et al. (1998) JMB 275:695-714; Chester et al., 2003 EMBO J. 22, 3971-3982).
(6) Editing Activity
209. Editing activity for wild type and mutant constructs of scCddl were measured as described previously and in the following examples. (7) Results
210. The hidden Markov modeling software SAM was trained with CDD 1 , APOBEC 1, APOBEC2, AID and phorbolin 1. This identified APOBEC3A, 3B, 3C, 3E, 3F, 3G, XP_092919, PHB1, XP_115170/XP_062365.
211. PHI-BLAST, using the target pattern H[VA]-E-x-x-F-(x) 19-[17V]-[T/V]- [W/C]-x-x-S-W-[ST]-P-C-x-x-C (SEQ ID NO: 60) limited the search more and misses only the 3B (Phorbolin 2) variant AAD00089 in which a single codon change GAC/T (SEQ ID NO: 63) to GAA/G (SEQ ID NO: 64) changes the ZDD center HxE to HxA. This is either a sequencing enor or a significant SNP for psoriasis.
212. [HC]-x-E-x-x-F-x(19,30)-P-C-x(2,4)-C (SEQ ED NO: 61) yields the usual suspects for human. There are a couple of novel deaminases with motif
HPE....SPC C. Also identifies a mouse gene homologous to hu APOBEC3G
(CEM15). On Chrom. 15, position 15E2. This is highly homologous to APOBEC3B, 05 0 '
Attorney Docket Number 21108.0035P1
D+E, G. There are 9 exons. Both ZDDs fall in their own exons. On the mouse gene, the start of the linker is an exon junction.
213. The multiple sequence alignment results are shown below in Table 4.
Table 4.
CLTJSTAL W (1.82) multiple sequence alignment
MouseAPOBEC3 -GPFCLGCSHRKCYSPIRNLISQE T|KFH|κ|LRY APOBEC3B -NPQIRNPMERMYRDTIYDNIEIEPI 25 MDS019 -KPHFRNTVERMYRDTISYNIYIRPI 25 APOBEC3F -KPHFRNTVERMYRDTISYNIYIRPI 25 D+E -NPQIRNPMERMYRDTIYDNIEIEPI 25 12g23newgene -KPQIRNLMEWMYPGT|YYN|E|RPI 25 APOBEC3A APOBEC3C NPQIRNPMKAMYPGT|YFQ|KI APOBEC3D NPQIRNPMERMYRDTIYDNIEIEPI 25 APOBEC3E NPMEAMYPHI|YFH|K|L-- APOBEC1 -TSEKGPSTGDPTLRRRIEPWEFDV|YDPRE 29 AID
APOBEC2 AQKEEAAVATEAASQNGEDLENLDDPEKLKE IELPPFEIVTGERLPANF|KFQ|R|VEY 60 XM 092919 SSKA LC SPGLPLPQPQP APSPLAPLPSP PPRLFALLNTMALLTAETIRLQINIKRR 60
Figure imgf000078_0001
MouseAPOBEC3 PENQQNiCRiVQEG AQVAA DLYE|KKBKKBDNGGRRFRPWKKLLTNFRYQDSK
APOBEC3B RDYRRAICRISQAG ARVTl|DYEE|AYβENBγNEGQQPMPWYKFDENYAFLHRT 185
MDS019 PDYQEAlRslcQKRDGPRATMKllNYDElQHHsKBYSQRELFEPWNNIiPKYYILLHIM 189
APOBEC3F RDYRRAICRISQAG ARVKIID-DEIAYHENHYSEGQPFMPWYKFDDNYAFLHRT 183
D+E RDWRWV| R|HKAG ARVKI|DYED|AYHENB|CNEGQP MPWYKFDDNYASLHRT 197
12q23ne gene ADYQEEf-- -
143
APOBEC3A --YKEA|QM|RDAG AQVSl|TYDE|KrβDϊβDHQG
APOBEC3C PCYQEG|RS|SQEG VAVEI|DYED|KYHENB|YNDN Attorney Docket Number 21108.0035P1
APOBEC3D RDWRWVHLREHKAG ARVKϊiDYEGER-
175
APOBEC3E TDYQEG|CS|SQEG- -ASVKI|GYKD|VSHKNB[YSDD
APOBEC1 QQNRQGIRDIVNSG- -VTIQI|R SE HBRNHNYPPGDE H- -132
AID
APOBEC2 PEIQ A. AIKKΪ KEAG- -CKLR II|j KPQD|EYV|QNB| [EE^QEEGE---
XM 092919 KPQQ :KKGGl|RL|I CGSQ- -VPVE:V|GFPK|ΆDHE:NBDΗ:EKPLSF-
10 MouseAPOBEC3 LQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRVHLLSEEEF
APOBEC3B KEILRY MD
MDS019 LGEILRHSMD
APOBEC3F 1KEILRNPMEAMY
D+E J-KEILRNPMEAMY
15 1 q23newgene HRWS- -LGS
APOBEC3A EASPASGPRH
APOBEC3C
APOBEC3D - CRGQGSMTGRNSLRDGWI CNAMAGQPAGVGLALIATDSQETRPGRAGPGSGES
APOBEC3E
20 APOBEC1
AID -DSLLMN
APOBEC2
XM 092919
25
MouseAPOBEC3 YSQLYNQRVKHLCYYHGMKPY II * QLEQFNGQAPLKG C LSEKGKQ
APOBEC3B PDT|TFN|N|DPLVLRRRQT IYY:EVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGR 242
MDS019 PPT|TFNINIEPWVRGRHET Y TE.VERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGR|246
APOBEC3F PHIIYFHIKILRKAYGRNESW FF1TMEVVKHHSPVSWKR- --GVFRNQVDPETHC 237
30 D+E PHI|YFH|K|LLKACGRNESW| FFTMEVTKHHSAVFRKR- --GVFRNQVDPETHC 251
12q23newgene TRPTCAADF 150
APOBEC3A LHl|TSN|N|GI- - -GRHKTYHfϊEVERLDNGTSVKMDQHRGFLHNQAKNL CGFYGR 9
APOBEC3C
APOBEC3D - 193
35 APOBEC3E
APOBEC1
AID RRK| YQ|κ|VRWAKGRRETYH'!^røRDSA'I'SFS ' -LDFGY RNKNGC|
APOBEC2
XM 092919
40
Figure imgf000079_0001
APOBEC3D 207
APOBEC3E
APOBEC1
AID V|LLHRYISDWD|DPGRC|R| |YD— HRHVAD|LRGNPNLS|R|FTARI>Y
55 APOBEC2 169
XM_092919
170
60
MouseAPOBEC3 FHWKRPFQ-KG|CS|WQSGILVDV|DLPQ|TDClTNβr—-
APOBEC3B DY- - DPLYKE7A|QMIRDAGAQVSI|TYDEIEYCIDTHYRQGC
MDS019 DD--QGRCQEG|RT|AEAGAKISIITYSE|KHC|DTHDHQGC
APOBEC3F YFWDTDYQ-EG|RslsQEGASVE|GYKDlκYclENβYNDDE
65 D+E YFWDTDYQ-EG|CS|SQEGASVKI|GYKD|VSCIKNHYSDDE
12q23newgene N YLPGYEG|CM| |vτH[DHQGC
APOBEC3A DY-DPL C
APOBEC3C E
APOBEC3D LSASHLGVPG FISD| YC|ENHCNEGQ
70 APOBEC3E E
Figure imgf000079_0002
Attorney Docket Number 21108.0035P1
Figure imgf000080_0002
Figure imgf000080_0001
The TBLASTN results are shown in Table 5:
Table 5
>gi|20902839|ref |XP 122858.l| (XM_122858) similar to hypothetical protein,
MGC:7002; hypothetical protein MGC7002 [Mus musculus] Length = 429
Score = 180 bits (457) , Expect = le-44
Identities = 47/171 (27%), Positives = 75/171 (43%), Gaps = 9/171 (5%)
Query: 14 LRRRIEPWEFDVFYDP RELRKEACLLYEIKW GMSRKIWRSSGKNTTN-HVEVNF 66
+R I F + + RK+ L YE+ + KN N H E+ F
Sbjct: 17 IRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICF 76
Query: 67 IKKFTS--ERDFHPSISCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMD 124
+ F + P ITW++SWSPC+EC++ I FL+ H ++L 1+ +RL+ D Sbjct: 77 LYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQD 136
Query: 125 QQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWM 175 (SEQ ID NO: 14)
+ +Q L LV G + M E+ CW+ FV+ W + + Sbjct: 137 PETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFR 187 (SEQ ID NO: 15)
Score = 121 bits (303), Expect = 8e-27
Identities = 41/168 (24%) , Positives = 71/168 (41%) , Gaps = 17/168 (10%)
Query: 16 RRIEP---WEFDVFYDPR ELRKEACLLYEIKWGMSRKIWRS--SGKNTTNHVE 63
RR++P EF + + R + L Y+++ + + + H E Sbjct: 231 RRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAE 290
Query: 64 VNFIKKFTSERDFHPSISCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHM 123
+ F+ K +IT +L+WSPC C+ + F P + L IY +RL++H
Sbjct: 291 ILFLDKI RSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHW 346
Query: 124 DQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYP 171 (SEQ ID NO: 16)
+ ++GL L SG+ + +M ++ CW NFV P W Sbjct: 347 KRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFV-NPKRPFWPWKGLE 393 (SEQ ID NO: 17)
>gi|l3384970|ref |NP 084531.1 | (NM_030255) hypothetical protein, MGC:7002; hypothetical protein Attorney Docket Number 21108.0035P1
MGC7002 [Mus musculus] qi j 13097063 | gb | AAH03314 .1 | AAH03314 (BC003314 ) Unknown (protein for MGC : 7002 ) [Mus musculus] Length = 429
Score = 176 bits (446) , Expect = 3e-43
Identities = 47/171 ( 27%) , Positives = 75/171 (43 %) , Gaps = 9/171 (5%)
Query : 14 LRRRIEPWEFDVFYDPREL RKEACLLYEIKW GMSRKIWRSSGKNTTN-HVEVNF 66 +R I F + RK+ L YE+ + KN N H E+ F
Sbjct: 17 IRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICF 76
Query: 67 IKKFTS--ERDFHPSISCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMD 124 + F + P ITW++SWSPC+EC++ + FL+ H ++L 1+ +RL+ D Sbjct: 77 LYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQVLRFLATHHNLSLDIFSSRLYNIRD 136
Query: 125 QQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWM 175 (SEQ ID NO: 18)
+N+Q L LV G + M E+ CW+ FV+ W + + Sbjct: 137 PENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFR 187 (SEQ ID NO: 19)
Score = 118 bits (297) , Expect = 5e-26
Identities = 37/165 (22%), Positives = 67/165 (40%), Gaps = 14/165 (8%)
Query 16 RRIEPWEFDVFYDPRELRK EACLLYEIKWGMSRKIWRS--SGKNTTNHVEVNF 66
+ EF + + ++ + L Y+++ + + + H E+ F Sbjct 234 HLLSEEEFYSQFYNQRVKHLCYYHGMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILF 293
Query 67 IKKFTSERDFHPSISCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQ 126
+ K IT +L+WSPC C+ + F P + L IY +RL++H + Sbjct 94 LDKI RSMELSQVIITCYDTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRP 349 Query 127 NRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYP 171 (SEQ ID NO: 20)
++GL L SG+ + +M ++ CW NFV P W Sbjct 350 FQKGLCSLWQSGILVDVMDLPQFTDCWTNFV-NPKRPFWPWKGLE 393 (SEQ ID NO: 21) The BLAST alignment is shown in Table 6:
Table 6 Score E
Sequences producing significant alignments: (bits) Value ref |NWJ00106.l|Mml5_WIFeb01_286 Mus musculus WGS supercont ... 1156 0.0
Alignments
>ref |NW_000106.l|Mml5_WIFeb01_286 Mus musculus WGS supercontig Mml5_WIFeb01_286 Length = 65562851
Score = 1156 bits (601), Expect = 0.0 Identities = 615/621 (99%), Gaps = 4/621 (0%) Strand = Plus / Plus
Query: 1223 agtcctggggtctgcaagatttggtgaatgactttggaaacctacagcttggacccccga 1282 Sbjct: 41563126 agtcctggggtctgcaagatttggtgaatgactttggaaacctacagcttggacccccga 41563185
Query: 1283 tgtcttgagaggcaagaagagattcaagaaggtcttttggtgacccccccacccaacccc ,
Attorney Docket Number 21108.0035P1
1 1111 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 11 11 1 1 1111 11 1 1 1 11 1 1 1 1 1 1 1 11 111 1 1 111 1 1 1 1 1
Sbjct : 41563186 tgtcttgagaggcaagaagagattcaagaaggtcttttggtgacccccccacccaacccc 41563245
5
Query: 1343 aagtctaggagaccttttgttctcccgtttgtttccccttttgttttatcttttgttgtt 1402
10 Sbjct: 41563246 aagtctaggagaccttttgttctcctgtttgtttccccttttgttttatcttttgttgtt 41563305
Query: 1403 ttgctttgttttgaagacagagtctcactgggtagcttgctactctggaactcactacta
Sbjct : 41563306 t 1t1g1c1t1t1t1g1t1t1t1t1g1a1a1g1a1c1a1g1a1g1t1c1t1c1a1c1t1g1g1g1t1a1g1c1t1t1g1c1t1a1c1t1c1t1g1g1a1a1c1t1c1a1c1t1aIcIt1a1 41563365
20
Query: 1463 gactaagctggccttaaactctaaaatccacctgccaatgccttctgagagccaggctta 1522
Sbj ct : 41563366 gactaagctggccttaaactctaaaatccacctgccagtgccttctgagagccaggctta 25 41563425
Query : 1523 aggtgtgcgctgcccactcccagccttaacccactgtggcttttccttcctctttctttt
30
Sbjct : 41563426 alglgltlgltlglclglcltlglclclclalcltlclclclalglclcltltlalalclclclalcltlgltlglglcltltltltlclcltltlclcltlcltltltlcltltltltl 41563485
35 Query: 1583 attatctttttatctcccctcaccctcccgccatcaataggtacttaattttgtacttga 1642
Sbjct : 41563486 attatctttttatctcccctcaccctcccgccatcaataggtacttaattttgtacttga 41563545
40
Query : 1643 aatttttaagttgggccaggcatggtggagcagcgtgcctctaatcgcaggcaggaggat 1702
45 Sbjct: 41563546 aatttttaagttgggccaggcatggtggagcagcgtgcctctaatcgcaggcaggaggat 41563605 y: 1703 ttccacgagcttgaggctagcctgatctacatagtgggctccaggacagccagaactaca
Figure imgf000082_0001
Sbjct: 41563606 tltlclclalclglalglcltltlglalglglcltlalglclcltlglaltlcltlalclaltlalgltlglglglcltlclclalglglalclalglclclalglalalcltlalclal 41563665
55
Query: 1763 cagagaccctgtctcaaaaataaatttagatagataaatacataaataaataaatggaag 1822
Sbjct : 41563666 cagagaccctgtctcaaaaataaatttagatagataaatacataaataaat ggaag
60 41563721
Query: 1823 aagtcaaagaaagaaagacaa 1843 (SEQ ID NO: 22)
1111111111111 II 111111
65 Sbjct: 41563722 aagtcaaagaaagaaagacaa 41563742 (SEQ ID NO: 23)
Score = 508 bits (264), Expect = e-141 Identities = 274/279 (98%) 70 Strand = Plus / Plus Attorney Docket Number 21108.0035P1
Query: 200 aggacaacatccacgctgaaatctgctttttatactggttccatgacaaagtactgaaag
5
Sbjct : 41553517 alglglalclalalclaltlclclalclglcltlglalalaltlcltlglcltltltltltlaltlalcltlglgltltlclclaltlglalclalalalgltlalcltlglalalalgl 41553576
10 Query: 260 tgctgtctccgagagaagagttcaagatcacctggtatatgtcctggagcccctgtttcg 319
Sbjct : 41553577 tgctgtctccgagagaagagttcaagatcacctggtatatgtcctggagcccctgtttcg 41553636
15
Query: 320 aatgtgcagagcaggtactaaggttcctggctacacaccacaacctgagcctggacatct 379
20 Sbjct: 41553637 aatgtgcagagcagatagtaaggttcctggctacacaccacaacctgagcctggacatct 41553696
Query: 380 tcagctcccgcctctacaacatacgggacccagaaaaccagcagaatctttgcaggctgg
Sbjct : 41553697 tlclalglcltlclclclglclcltlcltlalclalalclgtIaIcIaglglalclclclalglalalalcclclalglclalglalaltlcltltltlglclalglglcltlglgl 41553756
30
Query: 440 ttcaggaaggagcccaggtggctgccatggacctatacg 478 (SEQ ID NO: 24)
Sbjct: 41553757 t itmcagigiaiaigigiaigiciciciaigigitigigicitigicmcatigigianccitiaitiaicigi 41553795 (SEQ ID NO: 25)
35
Score = 502 bits (261) , Expect = e-139 Identities = 263/264 (99%) Strand = Plus / Plus
40
Query: 848 agaaaggcaaacagcatgcagaaatcctcttccttgataagattcggtccatggagctga
4 Λ5e S9b0j7ct: 41562163 alglalalalglglclalalalclalglclaltlglclalglalalaltlclcltlcltltlclcltltIgIaItlalalglaltltlclglgltlclclaltlglglalglcltlglal 41562222
Quer : 908 gccaagtgataatcacctgctacctcacctggagcccctgcccaaactgtgcctggcaac 50 967
Sbjct : 41562223 glclclalalgltlglalcaIaItIcIaIcIcItIgIcItIaIcIcItIcIaMccItIgMgaIgIcIcIcIcItIgMccIcIaIaIaIcItIgItIgIcIcItIgIgMcaIaIcI 41562282
55
Query: 968 tggcggcattcaaaagggatcgtccagatctaattctgcatatctacacctcccgcctgt 1027
Sbjc : 41562283 tggcggcattcaaaagggatcgtccagatctaattctgcatatctacacctcccgcctgt 60 41562342
Query: 1028 atttccactggaagaggcccttccagaaggggctgtgttctctgtggcaatcagggatcc
65
Sbjc : 41562343 a ItItMtcIcIaIcItIgIgIaIaIgIaIgIgIcIcIcItItIcIcIaIgIaIaIgIgIgIgIcItMgtIgItMtcItIcItIgItIgIgIcIaIaItIcIaIgIgIgMatIcIcI 41562402
70 Query: 1088 tggtggacgtcatggacctcccac llll (SEQ ID NO: 26) Attorney Docket Number 21108.0035P1
Sbjct: 41562403 tlglgltlglglalclgltlclaltlglglalclcltlclclclalcl 41562426 (SEQ ID NO: 27)
Score = 283 bits (147) , Expect = 2e-73 Identities = 155/159 (97%) Strand = Plus / Plus
Query: 691 aggcgagtgcacctgctaagtgaagaggaattttactcgcagttttacaaccaacgagtc 750
Sbjct : 41561266 aggcgaatggacccgctaagtgaagaggaattttactcgcagttttacaaccaacgagtc 41561325
Query: 751 aagcatctctgctactaccacggcatgaagccctatctatgctaccagctggagcagttc 810 Sbjct: 41561326 aagcatctctgctactaccaccgcatgaagccctatctatgctaccagctggagcagttc 41561385
Query: 811 aatggccaagcgccactcaaaggctgcctgctaagcgag 849 (SEQ ID NO: 28)
Sbjct: 41561386 aatggccaagcgccactcaaaggctgcctgctaagcgag 41561424 (SEQ ID NO: 29)
Score = 269 bits (140), Expect = 3e-69 Identities = 148/152 (97%) Strand = Plus / Plus
Query: 51 cagaaacctgatatctcaagaaacattcaaattccactttaagaacctacgctatgccat 110
Sbjct : 41551231 cagaaacctgatatctcaagaaacattcaagttccactttaagaacctaggctatgccaa 41551290
Query: 111 agaccggaaagataccttcttgtgctatgaagtgactagaaaggactgcgattcacccgt 170 Sbjct: 41551291 aggccggaaagataccttcttgtgctatgaagtgactagaaaggactgcgattcacccgt 41551350
Query: 171 ctcccttcaccatggggtctttaagaacaagg 202
Sbjct: 41551351 cltlclclcltltlclalclclaltlglglglgltlcltltltlalalglalalclalalglgl 41551382
Score = 212 bits (110) , Expect = 6e-52 Identities = 114/116 (98%) Strand = Plus / Plus
Query: 478 gaatttaaaaagtgttggaagaagtttgtggacaatggcggcaggcgattcaggccttgg 537
Sbjct: 41553934 gaatttaaaaagtgttggaagaagtttgtggacaatggtggcaggcgattcaggccttgg 41553993
Query: 538 aaaaaactgcttacaaattttagataccaggattctaagcttcaggagattctgag 593 (SEQ ID NO: 30)
Sbjct : 41553994 aaaagactgcttacaaattttagataccaggattctaagcttcaggagattctgag 41554049 Attorney Docket Number 21108.0035P1
(SEQ ID NO : 31 )
Score = 212 bits (110) , Expect = 6e-52 Identities = 112/113 {99% ) Strand = Plus / Plus
Query: 1112 agtttactgactgctggacaaactttgtgaacccgaaaaggccgttttggccatggaaag
Sbjct: 41562675 a 1g1t1t1t1a1c1t1g1a1c1t1g1c1t1g1g1a1c1a1a1a1c1t1t1t1g1t1g1a1a1c1c1c1g1a1a1a1a1g1g1c1c1g1t1t1t1t1g1g1c1c1a1t1g1g1a1a1a1g1 41562734
Query: 1172 gattggagataatcagcaggcgcacacaaaggcggctccacaggatcaaggag 1224
Sbjct: 41562735 glaltltlglglalglaltlalaltlclalglclalglglclglclalclalclalalalglglclglglcltlclclgclalglglaltlclalalglglalgl 41562787
Score = 187 bits (97) , Expect = 2e-44 Identities = 103/106 (97%) Strand = Plus / Plus
Query: 592 agaccttgctacatcccggtcccttccagctcttcatccactctgtcaaatatctgtcta 651 \
Sbjct : 41554842 agaccttgctacatctcggtcccttccagctcttcatccactctgtcaaatatctgtcta 41554901
Query: 652 acaaaaggtctcccagagacgaggttctgcgtggagggcaggcgag 697 (SEQ ID NO: 32) Sbjct: 41554902 acaaaaggtctcccagagacgaggttctgggtggagggcaggtgag 41554947 (SEQ ID NO: 33)
Score = 102 bits (53), Expect = 6e-19 Identities = 53/53 (100%) Strand = Plus / Plus
Query: 1 atgggaccattctgtctgggatgcagccatcgcaaatgctattcaccgatcag 53 SEQ ID NO: 34)
Sbjct: 41548340 a ItIgIgIgIaMccIaItItIcItIgItIcItIgIgIgIaItIgIcIaMgcIcIaItIcIgIcIaIaIaItIgIcItIaItItIcIaIcIcIgIaItIcIaIgI 41548392 (SEQ ID NO: 35)
3. Example 3 a) EXPERIMENTAL
214. All plasmids were constructed by standard recombinant DNA methods and verified by DNA sequencing. The intervening sequence (EVS)-apoB construct has been described previously (Sowden, M., et al. (1996) RNA 2, 274-288) Mutation of 6 bp at the 5 ' splice donor sequence, including the infronic GU dinucleotide (EVS- Δ5'apoB) and deletion of 20 bp encompassing the 3' splice acceptor and polypyrimidine tract sequences (TVS- Δ3'apoB), was accomplished by 'runaround' PCR using primers that included an Xhol site to facilitate subsequent re-ligation of the PCR Attorney Docket Number 21108.0035P 1
product (Fisher, C. L. et al. (1997) BioTechniques 23, 570-574.). IVS-Δ3'5'apoB was created by ligation of the appropriate halves of the above molecules.
215. McArdle RH7777 cells were maintained as previously described (Sowden, M.P. et al., (1996) J. Biol. Chem. 271 :3011-3017.) and fransfected in six-well clusters with 2 μg of DNA using lipofectAM NE® (Gibco BRL) according to the manufacturer's recommendations. RNAs were harvested 48 h post-fransfection in TriReagent (Molecular Research Center, Cincinnati, OH, U.S.A.) and subjected to reverse-transcriptase (RT)-PCR for amplification of intron-containing or exonic apoB specific transcripts using appropriate PCR primers as previously described (Sowden, M., et al. (1996) RNA 2, 274-288) and outlined in the Figure legends. Editing efficiencies were determined by poisoned-primer-extension assay on purified PCR products (Sowden, M., et al. (1996) RNA 2, 274-288) and quantified by analysis on a Phosphorlmager (model 425E; Molecular Dynamics).
216. The poisoned-primer-extension assay relies on the annealing of a 32P- end-labelled primer 3 ' of the editing site to the heat-denatured single-stranded PCR product. Extension of this primer using RT in the presence of dATP, dCTP, dTTP and dideoxy (dd)-GTP produces an extension product eight nucleotides longer if the cytidine has not been edited (CAA in the Figures); that is, incorporation of ddGTP causes chain tennination. If editing has created a uridine, then primer extension continues a further 11 nucleotides to the next 5' cytidine, where chain termination then occurs (UAA in the Figures). Quantification of the level of editing is accurately determined using laser scanning densitometry. The linear exposure range of the Phosphorlmager screen is sufficiently great to permit precise determination of low counts in the UAA bands whilst the high levels of counts in the CAA band remain in the linear range. Editing percentages were calculated as the counts in the UAA band divided by the total counts in the CAA plus UAA bands times 100. This assay has a lower level of detection of 0.1 %editing and remains linear up to 99.5%and is independent, between 1 ng and 500 ng, of the total amount of template PCR product used (M. P. Sowden, unpublished work). 217. Rev complementation/editing assays (Taagepera, S., et al. (1998) Proc.
Natl. Acad. Sci. U.S.A. 95:7457-7462.) were performed in duplicate in McArdle cells seeded in six-well clusters. Briefly, a total of 2 μg of DNA, comprising 1 μg of reporter 05 0
Attorney Docket Number 2 H0J 01)35P 1
DNA, 0.75 μg of transactivator DNA (pRc/CMV vector or a nucleocytoplasmic shuttling competent Rev-Rex fusion; a gift of Dr Thomas J. Hope, Infectious Disease Laboratory, Salk Institute for Biological Studies, La Jolla, CA, U.S.A.) and 0.25 μg of pRS V-p--galactosidase [internal confrol for chloramphenicol acetyl-fransferase (CAT) assays] were introduced into McArdle cells using lipofectAMINE® as described above. Cells were harvested at 48 h post-transfection, protein extracts prepared by freeze- thawing, and β-gal (Sowden, M.P., et al. (1989) Nucleic Acids Res. 17:2959-2972) and CAT (Neumann, J.R., et al., (1987) BioTechniques. 5:444-448.) assays performed as previously described. All extracts were nonnalized for b-gal activity. Parallel transfections were harvested for RNA preparation and RT-PCR amplification of the apoB RNA. Editing efficiencies were quantified as described above. b) RESULTS
(1) Introns interfere with editing 218. Previous studies demonstrated that the editing efficiency of apoB RNA was dramatically reduced when an infron was placed < 350 nt 5 ' or 3 ' of the target cytidine (Sowden, M., et al. (1996) RNA 2, 274-288). To provide proof that it was specifically RNA splicing and/or spliceosome assembly that had affected editing efficiency, splicing-competent and splicing-defective RNA transcripts were evaluated for their ability to support RNA editing in fransfected McArdle rat hepatoma cells. The apoB pre-mRNA reporter constract contained an abbreviated splicing cassette from the adenovirus late leader sequence fused to 450 nt of wild-type apoB mRNA (Figure 1A). Unspliced pre-mRNA and spliced mRNA were amplified from total cellular McArdle cell mRNA using the MS1/MS2 and SP6/T7 amplimer pairs respectively (Figure 1A). Consistent with previous results, the splicing cassette impaired the ability of the EVS- apoB RNA franscript to be edited, either before (pre-mRNA) or after (mRNA) it was spliced relative to a control transcript (pRc-apoB) that contained only apoB sequence (Figure IB). These results conoborate previous findings suggesting that there is a window of opportunity for editing apoB mRNA in the nucleus and that no further editing occurs in the cytoplasm of wild-type hepatic cells. Specifically, recently published subcellular-fractionation studies have shown that the low level of editing measured on this transcript as mRNA (1 %) occuned while the RNA was still in the nucleus (Yang, Y., et al. (2000) J. Biol. Chem. 275: 22663-22669.). Attorney Docket Number 21108.0035P1
219. Deletion of the polypyrimidine tract/branch point sequences and the 3 ' splice acceptor site in the EVS- Δ3'apoB franscript (Figure 1A) ablated the ability of this pre-mRNA to be spliced, as the SP6/T7 amplimer pair yielded only PCR products indicative of unspliced transcripts (results not shown). The editing efficiency of this splicing-defective constract was higher than that of TVS-apoB (14%, S.E.M=1.0 %; Figure IB). The EVS- Δ5'apoB franscript was also defective in splicing owing to deletion of the 5' splice donor sequence (the SP6/T7 amplimer pair failed to yield PCR products conesponding to spliced RNA; results not shown), and this RNA also demonstrated markedly elevated editing compared with EVS-apoB (11%, S.E.M.=0.1 %; Figure IB). The double-splice-site mutant EVSD3'5'apoB (Figure 1A) had an editing efficiency higher than either of the single-site mutants (20%, S.E.M.=0.2 %) and equivalent to the intron lacking RNA franscript, pRc-apoB (24%, S.E.M.=0.2 %; Figure IB). These results indicated that it is the assembly of a fully functional spliceosome and/or RNA splicing that impedes editosome assembly and/or function, and that both 5' and 3 ' splicing signals contribute to the inhibitory effect.
220. Each of the constructs in Figure 1 generated pre-mRNA transcripts of equivalent length, but the presence of active or inactive introns might influence expression levels of the resultant mRNAs. However, it was previously reported that the expression level of a given apoB transcript did not affect its editing efficiency (Sowden, M., et al. (1996) RNA 2, 274-288). Moreover, there was no competition between the editing efficiencies of exogenous and endogenous apoB transcripts, indicating that editing factors were not made to be rate-limiting by the increased concentration of apoB editing sites. These facts underscore the significance of the intron and RNA splicing on the regulation of editing efficiency. 221. In human aρoE3 mRNA, C6666 is located in the middle of the 7.5 kb exon
26, significantly further from a 5' or 3' intron than in the chimeric constructs described above. Therefore it was evaluated whether the proximity of the splice donor and acceptor sites to the tripartite motif affected editing efficiency. Insertion of a monomer or a dimer of the splicing-defective intron cassette (EVS Δ3'5') increased the distance between the active infron and the editing site by 425 and 850 nt respectively (Figure 2A). This increased the effective size of the chimeric exon to nearly 1 kb or 1.4 kb Attorney Docket Number 21108.0035P1
respectively; the average size of an internal exon being only 200-300 nt in mammals (Robberson, B.L., et al (1990) Mol. Cell. Biol. 10:1084-1094.).
222. ApoB pre-mRNA was amplified from each transcript expressed in McArdle cells using the MS7/MS2 amplimers and nesting with the MS2/MS3
5 amplimer pair. The sequence of primer MS7 is unique to the functional infron sequence and thus ensured amplification of unspliced pre-mRNA. Barely detectable levels of editing were measured on both pre-mRNA transcripts. However, a 10-fold higher level of editing was observed upon the spliced mRNA of both transcripts (6.0 %) (Figure 2B), which is 6-fold higher than the spliced mRNA derived from EVS-apoB (Figure
10 IB). This indicated that increasing the distance between the infron and the editing site alleviated, but was not completely capable of overcoming, the inhibitory effect of spliceosome assembly/RNA splicing on editing (i.e. compare 6 with 20% editing of EVS Δ3'5'apoB in Figure 1).
(2) The apoB editing site is not efficiently used
15 within an intron
223. A search of GenBank2 for apoB mooring-sequence similarities reveals numerous potential editing sites. However, many are located short distances from splice sites or within 5' or 3' untranslated regions or introns where the functional consequence(s) of a cytidine-to-uridine editing event is unclear. The release of the
20 entire human, mouse and rat genome sequences will likely reveal more mooring- sequence similarities, although their location in introns or exons may be uncertain until these genomes are annotated. In this regard, the results indicated that mooring- sequence-dependent editing sites may not be biologically active if they are positioned too close to splice junctions.
25 224. hi an attempt to be able to predict functional cytidine-to-uridine editing sites from these franscriptomes, it was investigated whether the apoB editing site is recognized when positioned within an intron. A 450 nt section of the apoB RNA franscript containing the editing site was placed within the infron of the adeno viras late leader sequence (IVS-apoB ENT) and this construct was expressed in transfected
30 McArdle cells. Pre-mRNA transcripts were amplified using the Exl/Ex2 amplimers followed by nested PCR with the MS Δ5/MSΔ6 amplimer pair and were edited at an efficiency of 0.4 % (Figure 3B). Intron-containing transcripts were amplified using the Attorney Docket Number 21108.0035P1
MS Δ5/MS Δ6 amplimers followed by nested PCR with the MS2/MS3 amplimer pair and were edited at an efficiency of 0.5 % (Figure 3B). The use of the MS Δ5/MS Δ6 amplimer pair in the initial PCR would not distinguish between unspliced pre-mRNA or spliced-out lariat RNA, but given the rapid degradation of lariat RNA, it is unlikely that the amplified PCR products represent lariat RNA species. If, however, there were amplified lariat species present, the difference of 0.1 % between infron-containing and unspliced pre-mRNA suggests that lariat RNAs containing apoB editing sites are not efficient editing substrates.
225. Mutation of the 5' and 3' splicing signals of the above construct to generate IVS- Δ3'5'apoB LNT restored editing efficiency (20%; Figure 3B) to a level equal to that of TVS- Δ3'5'apoB construct (20%; Figure IC). A minor additional primer extension product indicative of promiscuous editing was also apparent. These results support the hypothesis that pre-mRNA is not an effective subsfrate for cytidine-to- uridine editing and that this likely results from interference by spliceosome assembly/RNA splicing or potentially the rapid nuclear export of spliced mRNAs into the cytoplasm.
(3) Blocking the commitment of transcripts to the splicing pathway alleviates splice-site inhibition of editing 226. Most apoB mRNA editing subsfrate studies have employed cDNA transcripts which lack introns [(Sowden M.P., et al. (1998) Nucleic Acids Res. 26:1644-1652.; Driscoll, D.M., et al. (1993) Mol. Cell. Biol. 13:7288-7294.; Bostrom, K., et al. (1990) J. Biol. Chem. 265:22446-22452.)]. Wild-type apoB cDNA transcripts expressed in wild-type McArdle cells edit 2-3-fold more efficiently than the endogenous franscript (Sowden, M., et al. (1996) RNA 2, 274-288.; Sowden M.P., et al. (1998) Nucleic Acids Res. 26:1644-1652.). It has been demonstrated that chimeric splicing-editing reporter RNAs (EVS-apoB) had low editing efficiency as nuclear transcripts, which did not change once spliced mRNAs had entered the cytoplasm (Figure 1; (Yang, Y., et al. (2000) J. Biol. Chem. 275: 22663-22669.)). Hence the window of opportunity for a transcript to be edited in wild-type cells was confined to the nucleus, and when introns are proximal to the editing site, its utilization was impaired. Attorney Docket Number 21108.0035P1
227. To investigate if spliceosome assembly was involved in the inhibition of editing, and by-passing the spliceosome assembly commitment step inhibition may be alleviated (in a manner similar to intron-less cDNA transcripts), the processes of RNA splicing and RNA nuclear export were separated by utilizing a modification of the Rev complementation assay that has been employed to identify HEV-l Rev-like nuclear export sequences (Taagepera, S., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:7457- 7462.). Rev functions, by interaction with an RRE, to export unspliced RNA out of the nucleus. A reporter plasmid was constructed which contained an infron interrupted by the CAT gene and a functional apoB RNA editing cassette (Figure 4A). CAT activity could only be expressed if unspliced RNA was exported to the cytoplasm, a process wholly dependent upon an active Rev protein expressed from a co-fransfected plasmid. In the presence of Rev, spliceosome assembly on the transcript does not occur and therefore should not interfere with the utilization of the apoB editing site contained with the intron. 228. McArdle cells were co-fransfected with the modified reporter construct, together with either a control vector or a Rev expression vector. CAT activity was determined 48 h later (Figure 4B). In the presence of the control vector, very low levels of CAT activity were expressed, presumed to be due to splicing and degradation of the CAT transcript as a lariat RNA. Expression of the Rev protein resulted in nuclear export of unspliced infronic RNA and translation of the CAT protein, as evident in the 7-fold higher level of CAT activity in these cell extracts. These findings demonstrated that, in McArdle cells, HIV-1 Rev protein successfully diverted RNAs from the spliceosome assembly pathway and transported them into the cytoplasm.
229. Total cellular RNA was harvested from parallel transfections, the apoB sequence amplified, and the editing efficiencies were determined (Figure 4C).
Consistent with the findings described above, editing of apoB RNA within an infron of the RRE constract in the absence of Rev expression was very low ('infron+exon' amplified with EF/MS2). However, the editing efficiency was enhanced 5-fold when the Rev protein was co-expressed. Given that editing in the cytoplasm has never been demonstrated in wild-type McArdle cells (Yang, Y., et al. (2000) J. Biol. Chem. 275: 22663-22669.), nor would it be driven by an increase in apoB RNA abundance in the cytoplasm (Sowden, M., et al. (1996) RNA 2, 274-288), it appears enhanced editing Attorney Docket Number 21108.0035P1
occuned in the nucleus as a consequence of pre-mRNAs by-passing commitment to the spliceosome assembly and/or RNA export pathways. Editing unspliced CAT-apoB chimeric RNAs in the cytoplasm necessitates the activation of cytoplasmically localized editing factors by Rev. 5 230. In addition to an enhanced editing efficiency, the unspliced CAT-apoB
RNA was also promiscuously edited (additional primer extension stop labeled ' 1 ', Figure 4C). Promiscuous editing does not occur under physiological expression levels of APOBEC-1 in McArdle cells (Sowden, M., et al. (1996) RNA 2, 274-288.; Sowden, M.P. et al, (1996) J. Biol. Chem. 271:3011-3017.; Siddiqui, J.F., et al. (1999) Exp Cell
10 Res. 252: 154-164.), in rat tissues or under biological conditions where editing efficiencies are greater than 90%, e.g. rat intestine (Greeve, J., et al. (1993) J. Lipid Res. 34:1367-1383.). Nor does it occur when rat hepatic editing efficiencies are stimulated by metabolic or hormonal manipulations (Lau, P.P., et al. (1995) J. Lipid Res. 36:2069- 2078.; Baum, C.L.. et al. (1990) J. Biol. Chem. 265: 19263-19270.). Promiscuous
15 editing appears to be unique to cells in which APOBEC-1 has been artificially overexpressed (Sowden, M., et al. (1996) RNA 2, 274-288.; Sowden, M.P. et al., (1996) J. Biol. Chem. 271:3011-3017.; Siddiqui, J.F., et al. (1999) Exp Cell Res. 252:154-164.) and is observed under these conditions on both nuclear and cytoplasmic transcripts (Yang, Y., et al. (2000) J. Biol. Chem. 275: 22663-22669.). The results
20 presented in Figures 3 and 4 are therefore the first demonstration of promiscuous editing in the nucleus without the exogenous over-expression of APOBEC-1. c) DISCUSSION 231. ApoB mRNA editing, while conceptually a simple process of hydrolytic cytidine deamination to uridine (Johnson, D.F., et al. (1993) Biochem. Biophys. Res.
25 Commun. 195 : 1204- 1210.) has complexities in both the number of proteins involved and the cell biology involved in its regulation. It is well established that a sequence element consisting of three proximal components (enhancer, spacer and mooring sequence) comprise the 's-acting sequences required for efficient site-specific editing of C6666 in apoB mRNA Smith, H.C., et al (1991) Proc. Natl. Acad. Sci. U.S.A.
30 88:1489-1493; Backus, J.W., et al, (1992) Nucleic Acids Res. 20: 6007-6014; Smith, H.C. (1993) Semin. Cell. Biol. 4:267-278; Shah R.R., et al. (1991) J. Biol. Chem. 266:16301-16304; Backus, J.W., et al, (1991) Nucleic Acids Res. 19: 6781-6786; Attorney Docket Number 21108.0035P1
Driscoll, D.M., et al. (1993) Mol. Cell. Biol. 13: 7288-7294.). A multiple protein editosome catalyses and regulates editing of C [Smith, H.C., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:1489-1493; Harris, S.G., et al. (1993) J. Biol. Chem. 268:7382- 7392; Yang, Y., et al. (1997) J. Biol. Chem. 272: 27700-27706.). The components of 5 the minimal editosome from defined in vitro system analyses are APOBEC-1 as a homodimeric cytidine deaminase (Lau, P.P., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91 :8522-8526.) bound to the auxiliary protein ACF/ASP that serves as the editing-site recognition factor tlirough its mooring-sequence-selective RNA-binding activity (Mehta, A., et al. (2000) Mol. Cell. Biol. 20:1846-1854; Lellek, H., et al. (2000) J. Biol.
10 Chem. 275 : 19848- 19856.). Several other auxiliary protein candidates have also been described that had binding affinities for APOBEC-1 and/or apoB mRNA and that demonstrated the ability to modulate editing efficiency (Giannoni, F., et al. (1994) J. Biol. Chem. 269:5932-5936;Ymanaka, S., et al. (1994) J. Biol. Chem. 269:21725- 21734; Yang, Y., et al. (1997) J. Biol. Chem. 272: 27700-27706; Lellek, H., et al.
15 (2000) J. Biol. Chem. 275:19848-19856; Teng, B., et al. (1993) Science 260:1816- 1819; Inui, Y., et al. (1994) J. Lipid Res. 35:1477-1489; Anant, S.G., et al. (1997) Nucleic Acids Symp. Ser. 36:115-118; Lau, P.P., et al. (1997) J. Biol. Chem. 272:1452- 1455.). Although, under biological conditions, editing occurs only in the nucleus (Lau, P.P., et al. (1991) J. Biol. Chem. 266, 20550-20554; Yang, Y., et al. (2000) J. Biol.
20 Chem. 275:22663-22669.), nuclear and cytoplasmic distributions have been described for both APOBEC-1 and ACF (Yang, Y., et al. (2000) J. Biol. Chem. 275:22663- 22669; Yang, Y., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:13075-13080; Dance, G.S.C., et al. (2000) Nucleic Acids Res. 28:424-429.). Nuclear editing has been characterized as occuning coincident with, or immediately after, pre-mRNA splicing
25 (Lau, P.P., et al. (1991) J. Biol. Chem. 266, 20550-20554; Yang, Y., et al. (2000) J. Biol. Chem. 275:22663-22669; Sowden, M., et al. (1996) RNA 2:274-288.). Prior to splicing, pre-mRNA was not efficiently edited (Lau, P.P., et al. (1991) J. Biol. Chem. 266, 20550-20554.). It was not apparent, given the size of exon 26 and the nature of the cts-acting RNA sequence requirements, why there was a lag in editing activity during
30 pre-mRNA maturation. This question was addressed in studies indicating that spliceosome assembly and/or nuclear RNA export pathways regulate the utilization of cytidine-to-uridine editing sites. Attorney Docket Number 21108.0035P1
232. hi reporter RNA constracts, introns within 350-1000 nt of the apoB editing site suppressed editing efficiency. This inhibition was dependent on an active 5' splice site and/or 3' splice donor site and was partially alleviated after the reporter RNA had been spliced. This indicates that the process of spliceosome assembly functionally interfered with editosome assembly and/or function. This is supported by the distance dependence of this inhibition. When the splice sites were located more distal to the editing site, editing efficiencies were increased albeit not to levels seen on RNAs that do not contain introns. The gating hypothesis (Sowden, M., et al. (1996) RNA 2, 274- 288) proposed that each apoB RNA had a temporal 'window of opportunity' to become edited during its splicing and export from the nucleus. In this model, factors involved in spliceosome and editosome assembly are thought to compete for access to the mRNA. Consequently it is predicted that there will be less steric hindrance between the spliceosome and the editosome, and editing efficiency will improve the more distal an infron is located relative to the editing site [e.g. EVS-(iVS Δ3'5')-apoB or EVS- (EVS Δ3 ' 5 ')2 -apoB compared with EVS-apoB] . This phenomenon might explain the lower editing efficiency of native apoB editing prior to splicing, because the native editing site is only three times further away from the 5' or 3' splice junctions than that used in our reporter RNA constracts.
233. Importantly, these results have implications for the prediction of novel mooring-sequence-dependent RNA-editing sites. Not only is there a requirement for a target cytidine to be appropriately located upstream of a mooring sequence, but for efficient utilization, the editing site should not be in close proximity to an intron. Considering that the average size of an internal exon is only 200-300 nt in mammals (Robberson, B.L., et al. (1990) Mol. Cell. Biol. 10, 1084-1094.), it is highly unlikely that a significant amount of mooring-sequence-dependent editing will be observed in mRNAs with standard sized exons. In fact an analysis of the human, mouse and rat expressed-sequence-tag databases by Hidden Markov modeling has confirmed that the majority of mooring-sequence identities within coding sequences are located proximal to intron/exon junctions. An evaluation of select RNA transcripts revealed that they were in fact not edited. Related to these observations are results showing that editing sites located within introns were not inefficiently utilized. Taken together, the results support the hypothesis that spliceosome assembly and editosome assembly processes Attorney Docket Number 21108.0035P1
are communicating a temporal and spatial relationship that ultimately determines the efficiency of mooring-sequence-dependent editing. Consistent with this communication between the spliceosome and editosome is the finding that several proteins that have a role in RNA structure and/or splicing have also been implicated in RNA editing as auxiliary factors. These include hnRNP C, hnRNP D, APOBEC- 1 -binding protein (which has homology with hnRNP A and B) and KSRP, a protein involved in alternative splice site utilization (Lellek, H., et al. (2000) J. Biol. Chem. 275:19848- 19856; Greeve, J., et al. (1998) J. Biol. Chem. 379:1063-1073; Anant, S.G., et al.
(1997) Nucleic Acids Symp. Ser. 36:115-118; Lau, P.P., et al. (1997) J. Biol. Chem. 272:1452-1455.).
234. The promiscuous editing observed on TVS- Δ3'5'apoB ENT was unexpected, given the nature of the franscript, i.e. a cDNA equivalent to EVS-
Δ3'5'apoB in Figure 1 on which no promiscuous editing was observed at equivalent editing at c6666. A possibility for this could be the fortuitous introduction of a pair of tandem UGAU sequences within the infronic sequence 3' of the editing site, a motif that has been previously shown to promote promiscuous editing (Sowden, M.P., et al.
(1998) Nucleic Acids Res. 26:1644-1652.).
235. The description of the relationship of RNA splicing and editing is unique for apoB cytidine-to-uridine mRNA editing. However, an emerging theme in RNA processing is an interdependence of multiple steps in RNA maturation. Perhaps the most relevant to apoB editing is the adenine-to-inosine editing of glutamate and 5- hydroxytryptamine receptors. In contrast with apoB mRNA editing, mRNA substrates that undergo adenine-to-inosine editing all require the presence of a complementary intron sequence to form a partially double-stranded RNA structure that is recognized by the appropriate ADAR1 or ADAR2 enzyme Simpson, L., et al. (1996) Annu. Re.
Neurosci. 19:27-52; Maas, S., et al. (1997) Cum. Opin. Cell. Biol. 9:343-349; Rueter, S. M. and Emeson, R. B. (1998) Modification and Editing of RNA (Grosjean, H. and Benne, R., eds.), pp. 343-361.) . The critical role of cts-acting infronic sequences indicates deamination is a nuclear event, and as the editing site is frequently located close to a 5' splice acceptor site (Higuchi, M., et al. (1993) Cell. 75:1361-1370;
Egebjerg, J., et al. (1994) Proc. Natl. Acad. Aci. U.S.A. 91:10270-10274.) suggests that the level of editing maybe influenced by interference or interaction with RNA splicing.
Figure imgf000096_0001
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For example, endogenously expressed GluR2 mRNA from neuronal cell lines is always edited to 100% at the Gln/Arg site, whereas unspliced GluR2 franscripts are edited to only 70-90% (Higuchi, M., et al. (1993) Cell. 75:1361-1370.), indicating a partial inhibition of splicing until editing has occuned. Conversely, the franscript of the Glu- 5 R6 gene contains three exonic editing sites (Ile/Val, Tyr/Cys and Gln/Arg) which are edited to different extents, indicating that there must be a tightly regulated and coordinated action of the appropriate ADAR and the spliceosome at each editing site (Kohler, M., et al. (1993) Neuron 10:491-500; Seeburg, P.H., et al. (1998) Brain Res. Rev. 26:217-229.). In crosses of ADAR2 +/- with GluR-B (R) +/+ mice, an influence
10 from the editing status of the Gln/Arg site on subsequent splicing of the downsfream infron was observed (Higuchi, M., et al. (2000) Nature 405:78-81.), indicating that these RNA processing events do not occur independently. The major steps in pre- mRNA processing, capping, splicing, 3 '-end cleavage and polyadenylation are coupled to transcription through recruitment of the necessary processing factors to the largest
15 subunit of the RNA polymerase H. This represents an efficient process for increasing local concentrations of related processing and transcription factors on pre-mRNAs as and when they are needed (Lewis, J.D., et al. (2000) Science 288:1385-1389.). Many analyses of RNA processing have attempted to identify active versus inactive populations of processing factors and have postulated that the greatest concentration of 0 factors may or may not conespond to sites of function, dependent upon metabolic activity (Spector, D. (1993) Annu. Rev. Cell. Biol. 9:265-315.). Specifically, recent photobleaching studies (Lewis, J.D., et al. (2000) Science 288:1385-1389. and references cited therein) suggested that 'speckles' conespond to sites where free small nuclear RNPs transiently assemble before recruitment by the C-terminal domain of 5 RNA polymerase H and transfer to nascent transcripts. It is easily conceivable, therefore, that the processes of RNA editing and RNA splicing should be tightly coordinated, and the observation of nuclear and cytoplasmically localized APOBEC-1 and ACF conesponds to active and inactive complexes respectively. These two components of the minimal editosome, together with other editosomal proteins if 0 necessary, could be rapidly recruited to newly synthesized apoB mRNA franscripts by a coordinated action of RNA polymerase π and spliceosome assembly. Attorney Docket Number 21108.0035P1
236. Most, if not all, known RNA processing reactions can occur in vitro, but they are not as efficient as in vivo. This is also true for in vitro apoB RNA editing reactions. However, TVS-apoB RNA transcripts were edited with the same efficiency as infron-less apoB transcripts in vitro. This indicates that the presence of an intron perse does not interfere with editing, but, as was shown, there is a clear interdependence of splicing and editing for editing site regulation and fidelity in vivo. Such interdependence is also exhibited in mammalian nonsense-mediated decay ('NMD') of RNA, wherein only RNAs that contain nonsense codons and that have passed through the spliceosome are 'marked' and targeted for decay (Le Hir, H., et al. (2000) EMBO J. 19:6860-6869.). This imprinting of nuclear pre-mRNA by proteins that remain bound in the cytoplasm is a means of mRNAs 'communicating their history' (Kataoka, N., et al. (2000) Mol. Cell. 6:673-682.) and/or perhaps ensuring that no further RNA processing/editing occurs in the cytoplasm (Maquat, L., et al. (2001) Ceel. 104:173- 176.). 237. In conclusion, it has been demonstrated a spatial and temporal relationship between RNA splicing and apoB RNA editing. The suppression of editing- site utilization by proximal introns can explain the uniquely large size of exon 26 and/or the scarcity of other mooring-sequence-dependent cytidine-to-uridine editing sites. Moreover, these studies highlight the need to consider apoB RNA editing as an integrated process with RNA transcription and splicing, potentially expanding the number of auxiliary factors that should be considered as involved in apoB RNA editing. 4. Example 4 a) Infectivity assay using CEM15/Vif 238. The infectivity assay was carried out in the context of Vif minus pseudotyped virases and 293 T cells either lacking or expressing CEM15. An assay was developed using VSV G-protein pseudotyped lentiviral particles that confirmed the inhibitory effect of CEMl 5 on the infectivity of vif+ and vif- HEV-l particles and is amenable to the rapid demarcation of the regions of HEV-l DNA (or RNA) that is the target for CEMl 5 catalytic activity. An Env-deleted HEV-l proviral DNA vector (derived from pNL43; AEDS Reagent Repository) was modified by replacement of Nef with a GFP reporter gene and two in-frame stop codons were inserted that abolished vif production (pHR-GFPΔVif) (confirmed by western blotting with anti- Attorney Docket Number 21108.0035P1
Vif antibodies (AEDs Reagent Repository). Stable, HA-tagged CEMl 5 expressing 293T cell lines were selected with puromycin and verified by western blotting with a HA specific monoclonal antibody (HA.11; BabCo) (Figure 11). The expression of similar levels of full-length HA-tagged CEMl 5 (or mutant derivative thereof) can be assayed as well. Although stractural modeling can predict focused mutations that impair deaminase activity without destabilizing the entire protein, expression of the mutants should be verified. The addition of the HA epitope tag has no effect on the ability of CEM15 to suppress infectivity (Sheehy et al. Nature 418:646-650, 2002). Isogenic HEV-l pro-viral DNAs will be packaged into pseudotyped lentiviral particles by co-transfection with a plasmid encoding the VS V G-protein into 293T cells that lack endogenous CEM15 (-) or expressed wild type CEM15 (+) (Figure 11). The resulting pseudotyped particles contain HEV-l RNA of near full-length (with only a ~2kb deletion) were quantified by reverse transcriptase (RT) assay. p24Gag protein content can also be assayed by ELISA to normalize viral particles. A defined number (lxlO5 cpm of RT activity) of these particles were added to target, viras susceptible
MT2 cells (5x105). To assess their infectivity, the percentage of cells that expressed the GFP indicator gene encoded by the packaged recombinant HEV-l genome was quantified 24 hours later by flow cytometry (University of Rochester Core Facility). b) Results The results (Figure 12) indicate that the expression of CEMl 5 in 293T cells resulted in at least a 100-fold decrease in Vif- viral infectivity compared to particles generated in parental 293T cells. The low level of GFP expression from vif-, CEMl 5+ particles is indistinguishable from background fluorescence in confrol cells [0.2%]. 5. Example 5 Vif Antagonist Peptides
239. The cellular deaminase CEMl 5 can introduce multiple and therefore catastrophic dC to dU mutations in negative sfrand viral DNA following reverse transcription. This anti-viral activity is due to the inherent catalytic activity of CEMl 5 on single sfranded DNA and requires assembly of CEMl 5 within virions such that it is in position to interact with nascent cDNA during viral replication in the early stages of the HEV-l life cycle. Antiviral activity of CEMl 5, however, can be blocked by the viral .
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accessory protein known as viral infectivity factor or Vif (Sheehy et al. Nature 418: 646-650 (2002)).
240. Vif interacts with CEMl 5 and induces its poly-ubiquitination and degradation through the proteosome, thereby reducing the abundance of CEMl 5 and
5 promoting viral infectivity. It has been discovered that Vif homodimers were required for Vif s interaction with CEMl 5 (Yang et al. J Biol Chem. 278(8): 6596-602 (2003), US Patent 6,653,443, herein incorporated by reference in their entirety).
241. All peptides described above that block Vif s interaction with CEM15 and/or act to prevent CEMl 5 polyubiquitination have the effect of maintaining CEMl 5
10 intracellular abundance in viral infected cells. The effectiveness in the peptide Vif antagonist to block Vif and thereby protect CEMl 5 from degradation is reflected as a sustained abundance of CEMl 5 and this can be monitored by western blotting whole cell extracts and probing these blots with anti-CEM15 antibodies that are biologically relevant and is a rapid assay for VDA activity, ultimately V peptide Vif antagonist
15 activity. . Changes in viral infectivity can be determined by ELIS A quantification of HEV p24 antigen released from CEMl 5 positive cells that have been infected with wild type HEV-l and treated with or without peptide Vif antagonists. Western blotting for CEMl 5 can be conelated with peptide Vif antagonist protection of CEMl 5 with VDA suppression of viral infectivity. These studies can be performed over a range of peptide
20 Vif antagonist concentrations to establish a dose response relationship.
242. Commercially available services for high through put screening of chemical libraries can be used to identify small molecules that bind to the Vif dimerization domain peptide. These compounds can be tested for their ability to suppress CEM15 degradation and viral infected. CEMl 5, APOBEC-3F (h3F) and
25 possibly APOBEC-3B (h3B), previously refened to as Phorbolins, (Jarmuz et al., Genomics, 79(3):285-96 (2002)) are co-expressed in human lymphoid and myeloid cells, and as is the case for APOBEC-1, can form homodimers and also heterodimers (Bogerd et al., Proc Natl Acad Sci U S A 101(11):3770-4 (2004)). It has been shown that CEM15 and APOBEC-3F deaminate deoxycytidine on HEV-l and HEV-2 minus 0 strand cDNA. The dC to dU modifications template dG to dA mutations on the positive sfrand during replication, that inactivate multiple proteins essential for viral infectivity (Liu et al, J Virol, 78(4):2072-81 (2004). Zhang et al., Nature, 424(6944):94-8 (2003)). Attorney Docket Number 21108.0035P 1
Unlike APOBEC-1 and other ARPs, CEMl 5, APOBEC-3F and APOBEC-3B establish a close proximity with viral genomes, by becoming integrated within virions during their assembly (Stopak et al., Mol Cell, 12:591-601 (2003); Gaddis et al., J. Virol, 77(10):5810-5820 (2003); Mariani et al., Cell. 114(1):21-31 (2003); Wiegand, et al., Embo J, 23(12):2451-8 (2004)). With regard to the deaminase activity, dimers of deaminases such as APOBEC-1 and AJD are predicted to contain two catalytic centers (Xie et al., Proc Natl Acad Sci U S A, 101(21): 8114-9 (2004)). From structural modeling, it appears that in the dimer, a flexible flap domain from one catalytic center interacts with the other catalytic center and thereby regulates nucleic acid subsfrate binding. CEMl 5, APOBEC-3F and APOBEC-3B monomers each have two catalytic centers (both of which have activity (Mangeat et al., Nature, 424(6944): 99- 103 (2003); Shindo et al., J Biol Chem, (2003)). Homo- and heterodimers of CEMl 5, APOBEC-3F and APOBEC-3B therefore are predicted to have four catalytic centers and are likely to have considerable combinatorial substrate targeting potential that provides the host cell with an adaptive advantage against a broad spectrum of viruses.
243. HEV-l and HIV-2 use Vif to defeat the deaminase host defense. Vif has been shown to bind to both CEMl 5 and APOBEC-3F to target their ubiquitination and proteolytic degradation via the proteosome (Stopak et al. (2003); Mariani et al. (2003); Yu, X., et al. Science 302(5647): 1056-60 (2003); Zheng et al. J Virol. 78(l l):6073-6 (2004)). Vif s interaction with CEMl 5 occurs in a noncatalytic region that lies C- terminal to first catalytic domain. A single amino acid within this region (an aspartic acid in humans and a lysine in monkeys) provides the essential charge for the interaction of CEMl 5 with Vif (Bogerd et al. (2004); Mariani et al (2003), and Wiegand (2004)). Site-directed mutagenesis has shown that this single amino acid change in an ARP alters host range of a retroviruses (Bogerd et al. (2004), Mariani et al. (2003) and Xu et al., Proc Natl Acad Sci U S A, 101(15):5652-7 (2004). Due to this single amino acid difference simian viras (SEV) derived Vif cannot bind to human CEMl 5 and vise versa and consequently there is species-specific exclusion of CEMl 5 from the virion. Consequently, this region of CEMl 5 and APOBEC-3F can constrain the extent to which Vif can mutate and still protect the virus from the ARP-based host defense. .
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244. Vif forms homodimers, and Vif dimerization is required for viral infectivity. It has also been shown that Vif dimerization is required for Vif-dependent destruction of CEMl 5. Therefore, the Vif dimerization domain is a drug target for suppressing viral infectivity. HEV is notorious for its hypermutability and the acquired
5 resistance of this viras to therapy in AIDS patients. Vif has to interact with host cell CEMl 5 to protect the virus and therefore loss of Vif dimerization capacity through mutation may be less tolerated than are mutations in other viral proteins that have enabled the viras to acquire resistance to cunent therapeutic approaches. a) EXPERIMENTAL
10
245. CEMl 5 abundance can be quantified by western blotting as described above. Small molecules that bind to any of the aforementioned peptides can be evaluated for their ability to protect or restore CEMl 5 abundance using the aforementioned western blotting systems of whole cell extracts of cells that have been
15 transfected with CEM15 and Vif wherein these proteins are co-expressed in 293T cells (conditions that result in CEMl 5 destruction) and evaluated (by western blotting of cell extracts) for the ability of VDA peptides of varying size and sequence to restore CEM15 abundance. Co-expression of CEM15 and Vif by transfection in 293T cells results ~99% ablation of intracellular CEM15 within 36-48 h post-transfection.
20 Transduction of VDA into cells 6-12 hours following transfection results in restoration of CEM15. All peptides are tested according to this schedule. Expression of CEM15 and Vif are driven from the CMV promoter of pcDNA3 plasmids.
246. Determination of changes in endogenous CEMl 5 abundance. H9 cells express sufficient CEMl 5 that it is readily detectable by western blotting cell
25 extracts with monoclonal anti-CEM15 antibodies (4F11/Hl A, AIDS Research and Reference Reagent Program). This affords the opportunity to conelate viral infectivity measurements with endogenous CEMl 5 levels as the efficacy of optimized peptide Vif antagonists are evaluated in protecting endogenous CEMl 5 from Vif-dependent degradation. All assays of viral infectivity and the quantification of CEMl 5 are
30 performed in triplicate. Cells can be lysed and extracts blotted and reacted with antibodies as described above using the signal from GAPDH as a normalization value for comparing CEMl 5 levels between treatment groups. Attorney Docket Number 21108.0035P1
247. Small molecules that bind to the Vif dimerization domain and evaluate their ability to block Vif dimerization, prevent CEMl 5 degradation and suppression HEV-l infectivity. Peptides conesponding to the Vif dimerization can be used to screen chemical libraries for interacting compounds. 5 248. Analysis of the initial hits. The screen can yield numerous compounds.
Although the number of 'hits' can be greater had using full length Vif in the screening assay, probing the libraries with peptides containing Vif s dimerization domain selects for interactions that are more relevant to that domain and therefore compounds that are selected in this way stand the greatest possibility of having antiviral activity through
10 that mechanism.
249. Once interacting compounds have been identified, the initial evaluations can be done based on their ability to restore CEMl 5 abundance in Vif expressing cells using the western blotting assay described previously. This assay was chosen for the initial analysis of compounds over infectivity assays because given that CEMl 5
15 stability is widely accepted as a reliable predictor of viral infectivity, it is more rapid, cheaper and has a significantly lower biohazard risk. The screening nanows the pool of selected candidates from the initial screen to a half dozen or less compounds (SMVA candidates) for further validation. A dose range and time course in which maximum restoration of CEMl 5 abundance can occur can also be established.
20 250. These SMVA candidates then move on to secondary biological end point evaluations. This involves analysis of their ability to supress live virus infectivity as described above. Dose response curves can be established for all compounds that block viral infectivity.
251. Wash conditions varying in ionic strength, pH, detergent concenfration, 25 chaofropic agents or competitors are employed as a means to reduce nonspecific interaction and enrich for interactions with the highest specificity (lowest Kd). 6. Example 6
Reverse Transcription and Packaging Independent Antiviral Activity of CEM15 30 a) Summary
252. CEMl 5 (a.k.a. APOBEC-3G or h3G) functions as a natural defense against HEV-l viral infectivity by mutating the viral genome during its reverse
Figure imgf000103_0001
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transcription. This activity is inhibited by HEV-l viral infectivity factor (Vif) that is able to trigger degradation of CEMl 5 and prevent it from being packaged into the virion. However, this antiviral protein appears to have additional means by which it suppresses HEV-l. 253. Cells were fransfected with provirus DNA that produce pseudotyped viral particles in the absence of reverse transcription. CEM15 expression induced a marked (100-fold) reduction in viral particle production in the absence of Vif compared to that obtained from confrol cells or in the presence of Vif. This effect was due to a selective and marked reduction in viral protein and RNA. Reduction in viral particle production was also observed with a catalytically inactive mutant of CEM 15 showing that deaminase activity was not responsible for this antiviral mechanism. Vif expression blocked the effect of both CEM15 and the catalytic mutant CEM15 on viral production by inducing their degradation.
254. It was demonstrated that recombinant CEMl 5 can bind directly to RNA, which shows that it can play a role in the reduction of viral RNA. The phenotype described here differs from that in other reports in that it does not require CEMl 5 to become incorporated within virions or have mutagenic activity during reverse transcription. This mechanism can contribute important antiviral activity during late stages of the viral life cycle.
b) Introduction
255. Reverse transcription-dependent mutational activity of CEM15 on HEV- 1 ssDNA is not the only means by which CEM 15 can reduce viral infectivity. In fact, mutations in one or both of the zinc- dependent cytidine deaminase domains did not ablate CEM15's antiviral activity (Shindo et al., J Biol Chem (2003)). Moreover, blockage of reverse transcriptase (RT) processivity by CEMl 5 binding to the viral RNA templates has been suggested as an additional antiviral mechanism (Li et al., J Cell Biochem 92, 560-572 (2004)). In support of multiple mechanisms, transient expression of CEM 15 reduced the level of pseudotyped HEV-l particles generated from producer cells that were co- transfected with replication-defective proviral DNA constructs and helper plasmids (Sheehy et al., Nature 418, 646- 650 (2002)). This antiviral activity would have had to involve a mechanism that was independent of reverse transcription.
Figure imgf000104_0001
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256. It is shown that stably expressed CEM15 significantly reduced the level of pseudotyped HEV-l (particles lacking Vif. The reduced viral particle production is the result of a selective suppression of viral RNA leading to reduction in essential HEV- 1 proteins. These effects were not observed when Vif was expressed due to the marked reduction of CEM 15. Although CEM 15 was required to deplete viral particle production its deaminase function was not necessary. The data indicate an antiviral mechanism in producer cells which is potentially significant late during the viral life cycle that involves directly or indirectly the RNA binding ability of CEMl 5 and does not require virion incorporation of CEMl 5 nor viral replication. c) Experimental Procedures
257. Plasmid Constructions. CEMl 5 cDNA was RT-PCR amplified from oligo(dT)- primed total cellular RNA from CEM cells (Sheehy et al (2002). CEMl 5 deaminase domain mutations (DM) [E67A, E259A] were created by site-directed mutagenesis using the Quikchange system (Stratagene). Wild type CEMl 5 and DM were subcloned with an amino-terminal 6xHis and HA (hemagglutinin) tag into pfRES- P to permit CMV promoter driven expression of the cDNA and puromycin selection from an ECMV IRES element. pDHTV-GFP (from Dr. V. Planelles) is a pNL4-3 derived HIV-1 vector that contains a deletion of the env gene. pDHEV-GFP/ΔVif was constructed by inserting a 12bp fragment (5'-TAGTAACCCGGG-3', SEQ ID NO: 62) containing two termination codons underlined) at the PflMl site of pDHEV-GFP that lies near residue 89 of Vif, thereby leading to the production of a truncated and nonfunctional vif gene product. Cell culture and Transfection- 293T cells obtained from ATCC (Manassas, VA) were maintained in DMEM containing 10% fetal bovine serum plus penicillin/streptomycin/fungizone (Cellgro), and Non-Essential Amino Acids (Invitrogen) and were transfected using FuGENE 6 (Roche Molecular Biochemicals). Clonal cell lines were obtained by limiting dilution under 1 μg/ml puromycin selection.
258. Virus production. A two plasmid system was used to generate pseudotyped HEV-l particles. 293T cells stably expressing CEM15, DM, or empty pERES-P vector were fransfected with a mixture of pVSV-G and pDHEV-GFP (wt Vif) or pDHEV-GFP/Δ Vif using Lipofectamine 2000 (Invitrogen). Viruses were harvested at 48 and 72 hour post- transfection from culture supernatants and concentrated by ulfracentrifugation (22 K rpm,2 hour at 4oC). Attorney Docket Number 21108.0035P1
259. p24 and viral infectivity assays. Serial dilutions of viral stocks were assayed for p24 according to the manufacturer's recommendations (Beckman-Coulter, FL) and only results within the linear range of the standard curve were considered. Serially diluted viral stocks, normalized base on p24, were used to infect HeLa cells. 48
5 hours post- infection, cells were fixed and GFP expression analyzed by microscopy and flow cytometric analysis.
260. Cell lysates and western blot analysis. Cells were harvested by scraping into PBS containing a cocktail of protease inhibitors (0.5μg/mL each of aprotinin, pepstatin, and leupeptin, 1 mM PMSF (USB Corp), 2 mM Benzamidine and 2 mM
10 EGTA) at 24, 48, and 72 hours following transient transfection with HIV-1 plasmids. Cell pellets were lysed in Reporter lysis buffer (Promega) containing protease inhibitor cocktail. Protein concentrations were determined using the Bradford Assay (BioRad), and equivalent amounts of protein were analyzed by SDS-PAGE and subsequent western blotting using antibodies specific for HA (tagged CEM 15 and DM), D -actin,
15 and HEV-l RT (#6195), p24 (#287), Vif (#6459), Tat (#705) and Vpr (#3951) (Hauber et al., Proc Natl Acad Sci U S A 84, 6364-6368 (1987); Simon et al., J Virol 71:5259- 5267 (1997), Simon et al. J Virol 69:4166-4172 (1995), Fouchier et al. J Virol 70:8263- 8269 (1996)). Protein-RNA crosslinking. The indicated amounts of recombinant CEM15 (#10068, LmmunoDiagnostics, Inc., AEDS Reagent Repository) were added to
20 50 μl binding reactions containing 10 mM Hepes pH 7.9, 10% glycerol (v/v), 50 mM KCl, 50 mM EDTA, 0.25 mM DTT, 40 units of RNasin® (Promega), and 20 frnols of gel purified GP- RNA (nt 1573-2261; accession # K02013) or apoB RNA (nt 6413- 6860) (Smith, H.D., Methods 15:27-39 (1998)) that was 32P[ATP and CTP] labeled during in vitro T7 polymerase transcription (Promega). RNA binding reactions were 5 incubated at 30 oC for 3 h as previously described (Smith (1998)). Reactions were exposed to short wavelength ultraviolet (UV) light to induce protein-RNA crosslinking and subsequently digested with RNase A and Tl as previously described (Smith (1998)). Northern blot analysis. PolyA+ RNA prepared with a MicroPoly(A) Purist Kit (Ambion) according to manufacturer's protocol was resolved on a formaldehyde
30 agarose gel and transfened to nylon. The probe was GP-RNA cDNA radiolabeled with 32P[dCTP] using Ready-To-Go DNA labeling beads (Amersham Biosciences) according to the manufacturer's protocol. Blots were hybridized to the probe (lxlO6 Attorney Docket Number 21 108.0035P1
cpm ml) in ExpressHyb (Clontech) and washed according to the manufacturer's recommendations.
261. Blots were then stripped and reprobed with adenovirus E1A cDNA radiolabeled with 32P[dCTP] as stated above.
5 d) Results
262. To investigate alternative mechanisms that may contribute to the antiviral activity of CEMl 5, 293T cell lines stably expressing CEMl 5 (293T-CEM15) were selected and fransfected with plasmids containing replication-defective (Env- deleted) HEV-l proviruses (Vif+ or ΔVif) plus a helper/packaging plasmid (encoding
10 VSV-G). Culture supernatants from these cells were then assayed by p24 ELIS A, and a marked reduction of viral particle production (100-fold) by the ΔVif construct was detected in 293T- CEMl 5 versus the confrol, a 293T stable cell line containing pERES- P vector (Fig. 14). In contrast, Vif+ pro virus culture supernatants contained abundant viral particles, only 5- fold below control cells (Fig. 14). The infectivity of the
15 pseudotyped virus preparations was examined by transduction of HeLa cells with p24- normalized amounts of Vif+ and ΔVif virus particles. Consistent with prior reports (Shindo et al. (2003), Liu et al., J Virol 78:2072-2081 (2004), Mangeat et al. Nature 424:99-103 (2003)), the infectivity of ΔVif pseudotyped HEV-l particles was markedly reduced compared to the Vif+ viruses. The data demonstrated ΔVif viral particle 0 production could be significantly suppressed by CEM 15 expression, and that this effect could be overcome by Vif expression. Moreover the data indicated that there were two general effects of CEM 15 expression; one that manifests in reporter cells due to CEM 15 incorporation with virions and mutagenic deaminase activity during reverse transcription and a previously uncharacterized effect on viral particle production in 5 producer cells.
263. To evaluate whether the suppression of viral production was due to reduced viral protein abundance, following transfection with HEV-l proviral plasmid DNAs, cell lysates were prepared from an equivalent number of cells, normalized for the amount of protein, and evaluated by western blotting. CEM15 was expressed at 0 similar levels throughout the 72 hour period, however, the abundance of CEMl 5 was markedly decreased over the same time period in cells expressing functional Vif (Fig. 15A). These findings are consistent with the ability of Vif to target CEM15 for Attorney Docket Number 21108.0035P 1
proteolysis (Mariani et al. Cell 114:21-31 (2003);, Stopak et al. Mol Cell 12:591-601 (2003), Yu et al. Science 302:1056-1060 (2003)), but they also indicate that the level of CEM15 expression in our 293T stable cell lines is within a range that can be functionally suppressed by proviral Vif expression. 5 264. Consistent with the reduction in viral particle production (Fig. 14), a marked reduction in HEV-l p24 and RT protein was observed in 293T-CEM15 cells transfected with the Vif proviral DNA plasmids (compare Fig. 15A and C at 72 h). In contrast, 293T-CEM15 fransfected with Vif+ proviral DNA plasmid contained comparatively elevated levels of p24 and RT (Fig. 15A at 72 h). Similar effects were
10 also observed for the HEV-l regulatory protein, Tat, and the accessory protein, Vpr (Fig. 15A and C). These reductions in viral proteins were selective, since β-actin levels in the various lysates were virtually identical at all time points (Fig. 15A-C; note that lane- loading was normalized on the basis of total protein amount loaded). Furthermore, luciferase expression from a co-fransfected plasmid was also unaffected by CEM 15
15 expression confirming that CEMl 5 -mediated repression has viral specificity.
265. CEM 15 is predicted to contain two zinc-dependent deaminase domains (Wedekind et al. Trends Genet 19:207-216 (2003)), each of which has been shown to possess partial antiviral activity (Shindo et al. (2003)). Point mutations of the essential glutamate residue within each catalytic domain reduced significantly, but did not
20 abolish CEMl 5 -mediated inhibition of HEV-l infectivity (Mangeat et al. (2003)). To evaluate whether deaminase activity was required for the observed suppression of viral particle production, a 293 T cell line stably expressinglhe CEM 15 double mutant E67A/E259A (DM) was transfected with Vif+ or D Vif proviral DNA plasmids. As shown in Figure 14, expression of DM resulted in a strong inhibition of HEV-l particle
25 production in the absence of Vif (approx. 50-fold, compared to control cells). This suppression was roughly 2 to 2.5 fold weaker than that produced by wild-type CEMl 5, and could be overcome by expression of Vif (Vif+ virus). Consistent with this, expression of DM also reduced the levels of p24 and RT in the absence of Vif, although not to the same level as in 293T-CEM15 cells (compare Fig. 15A and B). Effects on
30 Tat and Vpr were somewhat more variable. These data suggested that a functional deaminase domain is important for the reduction in HEV-l particle production, but is not a requirement. In considering how CEM 15 might alter viral protein production, the Attorney Docket Number 21108.0035P1
possibility was evaluated that it might be acting on proviral plasmid DNA or viral RNA in the nucleus. Previous immunocytochemical analysis of HA-tagged CEMl 5 in 293T cells suggested a predominant if not exclusive cytoplasmic localization (Mangeat et al. (2003)). However, this observation does not preclude the possibility that, like 5 homologous proteins such as AED and APOBEC-1, CEM15 can shuttle between the nucleus and cytoplasm (Chester et al. Embo J 22:3971-3982 (2003), Yang et al., Exp Cell Res 267:153-164 (2001), Ito et al. Proc Natl Acad Sci U S A 101:1975-1980 (2004)). This kind of trafficking could permit CEMl 5 to act on double-stranded proviral plasmid DNA or on ss-plasmid DNA (during transcription), leading to
10 mutation and/or degradation of proviral template. This possibility was evaluated on proviral DNA isolated from 293T-CEM15 cells and control cells. No difference in DNA recovery was detected in 293T-CEM15 fransfected with ΔVif provirus compared to control cells transfected with 4-Vif provirus, and no dC to dU mutations in proviral DNA were evident as determined by uracil DNA glycosylase treatment of isolated viral
15 DNA and alkaline cleavage of apyrimidinic sites (Suspene et al. Nucleic Acids Res 32:2421-2429 (2004)). Thus, it was concluded that DNA mutational activity by CEM 15 in producer cells did not account for the reduced viral particle production.
266. It was also examined whether CEMl 5 might have the ability to selectively target the frameshift region in the viral Gag-Pol mRNA. This was of
20 interest in part because of the effect of CEMl 5 on the stability and proteolytic processing of the Gag precursor (Fig. 15 A), and also because of the Gag-Pol junction stem-loop stracture that is necessary for the minus one frameshift translation of Gag- Pol (Baril et al. Rna 9:1246-1253 (2003), Frankel et al. Annu Rev Biochem 67:1-25 (1998)). CEM15-dependent RNA editing activity or RNA binding activity as reported
25 for APOBEC-1 (MacGinnitie et al. 270:14768-14775 (1995), Anant et al.. Mol Cell Biol 20:1982-1992 (2000)) could disrupt secondary stracture or otherwise mutate coding capacity. To test for RNA editing, polyA+ RNA from 293T-CEM15 72 h post- transfection of ΔVif or Vif+ proviral plasmid DNAs was RT PCR amplified with primers for the Gag-Pol junction and protease region (GP-RNA, Fig. 16 A). 12 and 8
30 clones from ΔVif or Vif+ conditions (respectively) were sequenced and all were found to be identical to the original HIV-1 DNA, eliminating RNA editing of this region as a mechanism. Attorney Docket Number 21108.0035P1
267. CEMl 5 RNA binding capacity was determined in- vitro using purified recombinant CEMl 5 and radiolabeled RNA in our standardized ultraviolet light (UV) crosslinking assay (Smith, H.D. (1998), Galloway et al. 34: 24-526, 528, 530 (2003)). CEMl 5 bound to radiolabeled HEV-l GP-RNA in concenfration dependent manner
5 (Fig. 16B) however the yield of complexes was similar with an equivalent amount and specific activity of radiolabeled apoB mRNA containing the RNA editing site for APOBEC-1 (MacGinnitie et al. (1995) and Snant et al. (2000)). The nonselective interaction of CEM 15 with RNA is consistent with reports suggesting that RNA binding activity of CEMl 5 blocks RT progression on viral RNA (Li et al. (2004)) and 10 that its interactions with viral and cellular RNAs enable CEM 15 to assemble with virions (Svarovskaia et al. J Biol Chem (2004)). However, through the use of recombinant protein, it was established that CEMl 5 can bind to RNA in the absence of additional protein factors.
268. Considering the ability of CEMl 5 to interact with RNA, it was tested
15 whether it could modify the stability of the viral Gag-Pol mRNA. To evaluate mRNA stability, polyA RNA was collected from 293T-CEM15 at 24 h, 48 h and 72 h after transfection with proviral DNAs, and northern blot analysis was perfomied. The results revealed that viral RNA levels were depleted at all time points, in the absence of Vif (2- fold, 9-fold and 56- fold respectively, when compared to cell transfected with the Vif+
20 proviras) (Fig. 16C). CEMl 5 expression did not affect the abundance of an endogenous transcript present in 293 T cells (adeno viras El A RNA), as expected since luciferase and β-actin protein expression were also unaffected by CEMl 5. Hence El A RNA served as an internal loading control for comparison of viral RNA levels (Fig. 16C). Expression of the deaminase inactive DM also induced a depletion of viral RNA but to
25 a lesser extent (consistent with the recovery of viral proteins; Fig. 15B and 16C). Taken together with the aforementioned studies, these findings show that CEMl 5 binding to viral RNA alone or in conjunction with other viral or cellular proteins may have signaled for viral RNA degradation. e) Discussion
30 269. A considerable body of evidence indicates that the suppression of HEV-l infectivity by CEM 15 is due to a pleiotropic effect arising from its ssDNA mutating cytidine deaminase activity during viral RNA genome reverse transcription (Yu et al. Attorney Docket Number 21108.0035P 1
Nat Struct Mol Biol 11:435-442 (2004); Harris et al. Cell 113:803-809 (2003); Zhang et al. Nature 424, 94-98 (2003)). Studies in which either or both of the cytidine deaminase domains of CEMl 5 were mutated showed that both catalytic domains are functional in mutating HEV-l minus strand cDNA genomes (Shindo et al. (2003)). However, these studies also demonstrated partial suppression of viral infectivity by deaminase inactive CEMl 5. A role for CEMl 5 that does not involve ssDNA mutation has been suggested at the level of blocking the progression of reverse transcription on viral RNA templates (Li et al. (2004)). A novel mechanism was evaluated whereby CEM 15 suppressed HEV-l production, which does not depend on the incorporation of CEM 15 into the virion and/or viral reverse transcription. It was shown that CEM 15 selectively reduced viral RNA and protein abundance resulting in a phenotype of reduced viral particle assembly. This effect was not dependent upon CEM15-mediated DNA mutation or RNA editing and was largely abrogated by the expression of Vif. It ; was also revealed that recombinant CEM 15 can bind directly to viral Gag-Pol RNA and • non- viral RNAs. These findings conoborate recent reports of CEMl 5 's general RNA binding activity (Yu et al. (2004), Li et al. (2004), Svarovskaia et al. (2004)) and indicate that, either directly or indirectly, CEM 15 binding to viral RNA can lead to its premature decay, hi this regard, CEMl 5 interactions with Gag nucleocapsid (Cen et al. J Biol Chem (2004), Alee et al. J Biol Chem (2004)), and the ability of both proteins to bind HEV-l RNA can provide specificity resulting in the selective degradation of viral RNAs.
270. Previously, a significant impairment in viral production by CEMl 5- expressing 293T cells (Lin et al. (2004), Kao et al. J Virol 77:11398-11407 (2003)) has not been shown, but these experiments either relied upon a transient transfection of CEM15 (raising the possibility that some cells may have received HEV-l DNA in the absence of CEM15) (Kao et al. J Virol 77:11398-11407 (2003)) or they have involved stable co- expression of both CEM15 and proviral DNA (Lin et al. (2004)). hi the latter case, drug selection was used to establish the stable cell clones; which may have resulted in a powerful, positive selective pressure for rare cell clones in wliich hygromycin resistance gene (which was driven by the HEV-l LTR, and inserted into the pol region of the genome) was highly expressed. The ability to uncover the effect of Attorney Docket Number 21108.0035P 1
CEM 15 on viral RNA stability and protein production is therefore attributed to the fact that stable cell clones were used that uniformly express CEMl 5.
271. It was of interest that CEM 15 expression had a differential effect on viral protein abundance. The expression of the 55 kDa Gag precursor (p55) in proviral fransfected 293T-CEM15 cells was similar, regardless of whether Vif was expressed, but p24 abundance was markedly reduced in the absence of Vif (Fig. 15 A). The elevated levels of the ρ55 in 293T-CEM15 cells and DM cells fransfected with D Vif provirus, compared to confrol cells and DM cells fransfected with 4-Vif provirus (where p55 undergoes rapid and efficient cleavage) throughout the 72 hours suggested a lack of protease activity (compare Fig. 15A and B -Vif, contrast to B +Vif and C).
Furthermore, products of protease cleavage reactive with the RT-specific antibody were undetectable in 293 T- CEMl 5 cells in the absence of functional Vif (Fig. 15 A) (proteins detected included the product of initial protease cleavage [-116 kDa] (de Oliveira et al. J Virol 77:9422-9430 (2003)) and the fully processed RT heterodimer [p66 and p51] (Frankel et al. (1998), de Oliveira et al. (2003)). Collectively, these results suggested that functional protease activity in CEM 15 expressing cells was greatly diminished, possibly due to low amounts or the absence of the Gag-Pol precursor.
272. In conclusion, it appears that CEMl 5 can exert an antiviral effect during both the early and late phases of the HEV-l life cycle.
273. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.
274. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as Attorney Docket Number 21108.0035P1
exemplary only, with a trae scope and spirit of the invention being indicated by the following claims.
I ll Attorney Docket Number 21108.0035P1
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Claims

Attorney Docket Number 21108.0035P1CLAIMS
1. A method of screening for a Vif antagonist, comprising:
(a) contacting a Vif molecule with a test compound;
(b) detecting binding between the Vif molecule and the test compound; and
(c) screening the test compound that binds the Vif molecule for suppression of viral infectivity, suppression of viral infectivity by the test compound indicating the test compound is a Vif antagonist.
2. The method of claim 1 , wherein the viral infectivity is HEV-l infectivity.
3. The method of claim 1 , wherein the ability to suppress viral infectivity is measured by contacting the test compound with one or more cytidine deaminase- positive cells, in the presence of HEV-l viras expressing Vif .
4. The method of claim 3, wherein the cytidine deaminase-positive cells are CEM 15 positive cells.
5. A method of making a Vif antagonist, comprising:
(a) identifying the Vif antagonist of claim 1; and
(b) modifying the Vif antagonist to enhance suppression of viral infectivity.
6. A method of making a Vif antagonist, comprising :
(a) identifying the Vif antagonist of claim 1; and
(b) modifying the Vif antagonist to lower biotoxicity,
7. The method of claim 1 , wherein the Vif molecule is linked to a reporter.
8. The method of claim 7, wherein the reporter is luciferase.
9. The method of claim 7, wherein the reporter is GFP. Attorney Docket Number 21108.0035P1
10. The method of claim 7, wherein the reporter is RFP.
11. The method of claim 7, wherein the reporter is FITC.
12. The method of claim 7, wherein the Vif molecule and the reporter form a chimera.
13. The method of claim 7, wherein the Vif molecule comprises SEQ ED NO: 1.
14. The method of claim 13, wherein the Vif molecule has 80% or greater homology to SEQ ID NO: 1.
15. The method of claim 1, wherein a plurality of test compounds are contacted with Vif molecules in a high throughput assay system.
16. The method of claim 15, wherein the high throughput assay system comprises an immobilized anay of test compounds.
17. The method of claim 15, wherein the high throughput assay system comprises an immobilized anay of Vif molecules.
18. A Vif antagonist identified by the method of claim 1.
19. A Vif antagonist made by the method of claim 7.
20. A Vif antagonist made by the method of claim 8.
21. A method of screening for cytidine deaminase activators, comprising:
(a) contacting a cytidine deaminase molecule with a test compound;
(b) detecting binding between the cytidine deaminase molecule and the test compound;
(c) screening the test compound that binds the cytidine deaminase molecule to Attorney Docket Number 21108.0035P 1
identify a selected cytidine deaminase function, the presence of the selected function indicating a cytidine deaminase activator.
22. The method of claim 21, wherein the selected function of the cytidine deaminase is suppression of viral infectivity.
23. The method of claim 21, wherein the cytidine deaminase molecule is CEM15.
24. The method of claim 21, wherein the selected function of CEM15 is deoxycytidine mutation to deoxyuridine mutation in the first strand of cDNA of HEV-l during or subsequent to its synthesis by reverse transcriptase.
25. The method of claim 21 , wherein the selected function of CEM 15 is decreased , by binding to the test compound and cytidine to uridine editing of mRNA or deoxycytidine to deoxyuridine mutation of DNA is inhibited and associated cancer promoting activity or cancer phenotype is reduced.
26. The method of claim 21, wherein the cytidine deaminase molecule is APOBEC- 1.
27. The method of claim 21, wherein the cytidine deaminase activator is an APOBEC-1 activator.
28. The method of claim 26, wherein a selected function of APOBEC-1 is increased such that levels of apoB48 are increased due to cytidine to uridine editing of apoB mRNA and levels of apoBlOO are consequently decreased as compared to a control.
29. The method of claim 26, wherein a selected function of APOBEC-1 is decreased by binding to the test compound, and cytidine to uridine editing of mRNA or deoxycytidine to deoxyuridine mutation of DNA is inhibited, and associated cancer promoting activity is reduced.
Figure imgf000136_0001
Attorney Docket Number 21108.0035P1
30. The method of claim 21, wherein a selected function of the cytidine deaminase is promotion of antibody diversity produced by lymphocytes as compared to antibody production by confrol lymphocytes.
31. The method of claim 25, wherein the cytidine deaminase molecule is AED.
32. The method of claim 31, wherein a selected function of AED is increased such that levels of cytidine to uridine RNA editing or deoxycytidine to deoxyuridine mutation are increased and class switch recombination and somatic hypermuation within the immunoglobulin locus of genes within B lymphocytes is increased.
33. The method of claim 31, wherein a selected function of AED is decreased such that levels of cytidine to uridine RNA editing or deoxycytidine to deoxyuridine mutation are decreased and changes associated with cancer promoting activity are reduced
34. The method of claim 21, wherein the cytidine deaminase activator is an AED activator.
35. The method of claim 22, wherein the viral infectivity is HEV infectivity.
36. The method of claim 22, wherein ability to suppress viral infectivity is measured by contacting the test compound with a cytidine deaminase molecule in the presence of Vif and a viras.
37. A method of making a cytidine deaminase activator comprising:
(a) identifying the cytidine deaminase activator of claim 21; and
(b) modifying the cytidine deaminase activator to enhance the selected deaminase function of the modified cytidine deaminase activator as compared to the function of the unmodified cytidine deaminase activator. Attorney Docket Number 21108.0035P 1
38. A method of making a cytidine deaminase activator comprising:
(a) identifying the cytidine deaminase activator of claim 21; and
(b) modifying the cytidine deaminase activator to lower biotoxicity of the modified cytidine deaminase activator as compared to the biotoxicity of the unmodified cytidine deaminase activator.
39. The method of claim 21, wherein the cytidine deaminase molecule is linked to a reporter.
40. The method of claim 39, wherein the reporter is luciferase.
41. The method of claim 39, wherein the reporter is GFP.
42. The method of claim 39, wherein the reporter is RFP.
43. The method of claim 39, wherein the reporter is FITC.
44. The method of claim 39, wherein the cytidine deaminase molecule and the reporter form a chimera.
45. The method of claim 21, wherein the cytidine deaminase molecule comprises SEQ ED NO: 2.
46. The method of claim 41, wherein the cytidine deaminase molecule has 80% or greater homology to SEQ ID NO: 2.
47. The method of claim 21, wherein a plurality of test compounds are contacted with cytidine deaminase molecules in a high throughput assay system.
48. The method of claim 47, wherein the high throughput assay system comprises an immobilized anay of test compounds. Attorney Docket Number 21108.0035P1
49. The method of claim 47, wherein the high throughput assay system comprises an immobilized anay of cytidine deaminase molecules.
50. A cytidine deaminase activator identified by the method of claim 21.
51. A CEMl 5 activator identified by the method of claim 21.
52. An APOBEC-1 activator identified by the method of claim 21.
53. An AED activator identified by the method of claim 21.
54. A cytidine deaminase activator made by the method of claim 37.
55. A cytidine deaminase activator made by the method of claim 38.
56. A polypeptide comprising 5 or more contiguous amino acid residues of a ubiquitination protein, wherein the polypeptide binds Vif and blocks ubiquitination of CEM 15.
57. A polypeptide comprising 5 or more contiguous amino acid residues of a Gag protein, wherein the polypeptide binds CEM 15 and promotes CEM 15 binding to viral RNA.
58. A method of promoting CEMl 5 binding to viral RNA comprising contacting CEMl 5 with the polypeptide of claim 57.
59. A polypeptide comprising 5 or more contiguous amino acid residues of CEMl 5 wherien the polypeptide binds a ubiquitination protein and blocks Vif-mediated ubiquitination of CEM 15. Attorney Docket Number 21108.0035P1
60. A method of blocking the Vif-mediated ubiquitination of CEMl 5 comprising contacting the CEMl 5 with the polypeptide of claim 59.
61. A polypeptide comprising 5 or more contiguous amino acid residues of a CEMl 5 binding domain on Vif, wherein the polypeptide blocks CEMl 5-Vif interaction.
62. A method of blocking CEMl 5-Vif interaction comprising contacting Vif or CEMl 5 with the polypeptide of claim 61.
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