WO1997009345A1 - Virus protein purification from virosomes - Google Patents

Virus protein purification from virosomes Download PDF

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Publication number
WO1997009345A1
WO1997009345A1 PCT/US1996/014187 US9614187W WO9709345A1 WO 1997009345 A1 WO1997009345 A1 WO 1997009345A1 US 9614187 W US9614187 W US 9614187W WO 9709345 A1 WO9709345 A1 WO 9709345A1
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Prior art keywords
vap
ser
cleaved
thr
leu
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PCT/US1996/014187
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French (fr)
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Allen Portner
Toru Takimoto
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St. Jude Children's Research Hospital
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Priority to AU71545/96A priority Critical patent/AU7154596A/en
Publication of WO1997009345A1 publication Critical patent/WO1997009345A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention generally pertains to the field of viral protein crystallization.
  • the present invention specifically pertains to crystallization methods and crystallized viral attachment proteins (VAPs), obtained from virosomes.
  • VAPs crystallized viral attachment proteins
  • the crystallized VAP is biologically active.
  • Crystallized VAP, nucleic acid, vectors and host cells of a strain of paramyxovirus are also provided.
  • viruses contain an inner virion core having nucleic acid and a lipid envelope which holds the transmembrane (hydrophobic) domains of the envelope proteins.
  • Some of the envelope proteins are viral attachment proteins (VAPs) that contain extracellular domains. Viruses infect a target cell by association ofthe virus' VAP with the target cell's viral receptor.
  • VAPs viral attachment proteins
  • the extracellular domain ofthe VAP binds the target cell receptor and the transmembrane domain anchors the VAP to the viral envelope. (White et al, Quant. Rev. Biophys. 16: 151-195 (1983)). After association of the VAP with the cell's viral receptor, the virion core enters the cytoplasm ofthe bound cell and the viral replication process is initiated. In some cases, viruses that are bound to target cell receptors can enter the cells by receptor mediated endocytosis. Infectious Envelope-Containing Viruses
  • infectious envelope-containing viruses include, but are not limited to, togaviruses (yellow fever, RSSE and rubella); retroviruses (leukemia, sarcomas); orthomyxoviruses (influenza A, B, C); paramyxoviruses (mumps, measles, paramfluenza, Newcastle disease); rhabdoviruses (rabies); hepatitis virus; herpes viruses (herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr); and poxviruses (varcola, vaccinia, Molluscum Contagiosum). See, e.g., Lycke andNorrby, eds. Textbook of Medical Virology, Chs. 1-4 and 7-9, Butterworths, London (1983).
  • Paramyxoviruses are one ofthree genera ofthe family Paramyxoviridae, which includes enveloped, negative-stranded RNA viruses. Paramyxoviruses utilize hemagglutinin neuraminidase (HN) as the target VAP (Fraenkel-Conrat and Wagner, eds., Comprehensive Virology, Vol. 4,
  • paramyxoviruses include mumps, measles, parainfluenza virus (PIV), Sendai virus (SV) and Newcastle disease virus (NDV).
  • Electron micrographs have demonstrated that all paramyxovirus species have the same basic morphology.
  • This morphology includes highly pleomorphic particles that are enclosed by a lipid envelope acquired during maturation. Maturation usually occurs by virus budding from the plasma membrane ofthe host cell.
  • the viral membrane of a paramyxovirus contains two virus-specified glycoproteins, HN and F.
  • HN and F are found in all strains of paramyxoviruses.
  • HN and F are attached to the viral membrane by short N-terminal and C-te ⁇ ninal transmembrane peptide sequences, respectively.
  • the nucleotide sequence of HN genes of several paramyxoviruses has been determined. See, e.g., Gorman etal, Virology 775:211-221 (1990); Merson etal, Virology 167:91-105 (1988)); Blumberg et al., Cell 41:269-218 (1985); Paterson et al, Proc. Natl. Acad. Sci.
  • HN and F glycoproteins from different paramyxovirus strains exhibit the same biological activities.
  • Hemagglutination activity is the capacity of a virus to absorb to erythrocytes and, as a result, cause the erythrocytes to aggregate (agglutinate).
  • a protein projecting from the virus membrane surface (HN) mediates the attachment to a sialic acid glyco- conjugate receptors on the erythrocyte surface.
  • the hemagglutination reaction (HA) is an example of a relatively simple, quick, convenient and semi-quantitative way of detecting, identifying, titrating viruses, detecting viral antibody and studying virus attachment.
  • Cell-binding activity is the capacity ofa virus to attach to a variety of infectible host cells.
  • the HN protein of paramyxoviruses mediates the attachments to host cells, via a sialic acid-containing glyco-conjugate receptor.
  • Neuraminidase activity is the enzyme catalyzed cleavage of the ⁇ -ketosidic linkage between terminal sialic acid and an adjacent sugar residue.
  • the HN protein of paramyxoviruses possess neuraminidase activity. Fusion promoting activity is the capacity of paramyxovirus VAPs (e.g., HN, H) to provide an essential function that allows the fusion (F) protein to directly mediate virus host-cell and cell-to-cell membrane fusion. (Fraenkel-Conrat, supra, pp.
  • HN cell-binding, neuraminidase, and fusion promoting activities, essential for virus infection and spreading, are conserved among all or most strains of paramyxoviruses, as reflected in the high degree of sequence identity among these proteins. Therefore, the determined three-dimensional structure of an HN from a strain of paramyxovirus is useful for rational design of inhibitors to treat infections of many or most paramyxoviruses and may be applicable to other members of Paramyxoviridae family.
  • the crystal structures of neuramimdases, from influenza virus, Salmonella tryphimurium, and Vibrio cholerae show similar three-dimensional structures (Crennell et al, Structure 2:535-544 (1994); Crennell et al, Proc. Natl. Acad. Sci. USA 0:9852-9856 (1993)).
  • the present invention provides methods of purifying and crystallizing a viral attachment protein (VAP) from an envelope containing virus.
  • VAP viral attachment protein
  • the present invention also provides crystallized VAP which is soluble and biologically active.
  • the present invention also provides antibodies specific for the VAP and host cells that produce the antibody.
  • the present invention further provides nucleic acid molecules encoding the VAP, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the molecule.
  • the present invention also provides a crystallized HN protein from a strain ofa species of a paramyxovirus.
  • the present invention also provides antibodies specific for the HN and host cells that produce the antibody.
  • the present invention further provides nucleic acid molecules encoding the HN, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the nucleic acid.
  • the present invention also provides a crystallized HN protein from the Kansas strain of a species of a paramyxovirus: Newcastle disease virus (NDV).
  • This HN crystallized protein is suitable for x-ray diffraction analysis.
  • the x-ray diffraction patterns obtained by this analysis provide coordinates of moderately high to high resolution. These coordinates are useful for three dimensional modeling of the HN protein.
  • the three dimensional modeling programs use these coordinates and the amino acid sequence to generate secondary, tertiary and quaternary structures ofthe Kansas NDV HN.
  • the present invention also provides antibodies specific for the Kansas NDV HN and antibody expressing host cells.
  • the present invention further provides nucleic acid molecules encoding the Kansas NDV HN, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the nucleic acid.
  • FIG. 3 A diffraction pattern is presented from a crystal of Kansas strain NDV cleaved HN using an X-ray source. The resolution was 3.5 A at the edge ofthe pattern.
  • Figure 4 A diffraction pattern is presented from a crystal of cleaved HN using a more powerful X-ray beam than in Figure 3, produced in synchrotron storage rings. The resolution ofthe pattern was 2.6 A at the edge. Data was collected using a crystal frozen at -175 °C.
  • FIG. 1 The nucleotide sequence of the Kansas strain of NDV is presented.
  • Figure 6. The deduced amino acid sequence of an HN ofthe Kansas strain of NDV is presented.
  • the present invention overcomes one or more deficiencies ofthe related background art, by providing methods for crystallizing a viral attachment protein (VAP) from virosomes, where the crystals diffract x-rays with high resolution of 1.5-3.9A, such as 2.4-27A.
  • VAP viral attachment protein
  • the present invention thus includes methods of purifying and crystallizing a VAP from virosomes derived from a virus.
  • the present invention also provides crystallized VAP by these methods which is soluble and biologically active.
  • the present invention in a non-limiting example, provides methods of purifying and crystallizing hemagglutinin neuraminidase (HN) from a strain of a paramyxovirus using virosomes.
  • HN hemagglutinin neuraminidase
  • the present invention also provides crystallized HN by these methods which is soluble and biologically active.
  • the present invention also provides biologically active VAPs.
  • a non-limiting example is an HN from the Kansas strain of a species ofa paramyxovirus, the Newcastle disease virus (NDV).
  • the VAP is also provided as a crystallized protein.
  • a VAP from a virus is isolated in soluble form (e.g., lacking the transmembrane domains) by cleavage employing a protease applied to purified viruses or virosomes, as described herein.
  • the resulting cleaved VAP is in sufficient purity and concentration (e.g., a monomer or dimer) for crystallization.
  • the cleaved VAP is then isolated and assayed for biological activity and for lack of aggregation (which interferes with crystallization).
  • the purified and cleaved VAP preferably runs as a single band under reducing or nonreducing polyacrylamide gel electrophoresis (PAGE) (nonreducing is used to evaluate the presence of cysteine bridges).
  • PAGE polyacrylamide gel electrophoresis
  • the purified cleaved VAP is preferably crystallized using the hanging drop method under varying conditions of at least one ofthe following: pH, buffer type, buffer concentration, salt type, -o-
  • crystallized protein is also tested for neuraminidase or cell binding biological activity and differently sized and shaped crystals are further tested for suitability for X-ray diffraction. Generally, larger crystals provide better crystallography than smaller crystals, and thicker crystals provide better crystallography than thinner crystals.
  • a strain of virus is diluted in a buffer solution at about neutral pH.
  • the diluted virus solution can also be inoculated into the allantoic cavity of embryonated hen eggs for amplification.
  • Tissue culture of a virus strain, or recombinant expression ofthe VAP can alternatively be used according to known method steps.
  • infected, embroynated eggs are incubated for several days and then chilled at about 4°C or less overnight.
  • the allantoic fluids are collected and centrifuged at about 4°C or less to remove red blood cells.
  • the virus in the supernatant is sedimented by ultracentrifugation at about 4°C or less. After the virus pellet is soaked in buffer solution overnight at about 4°C or less, the pellet is resuspended, e.g., by homogenization.
  • the resuspended virus is optionally further purified by centrifugation in a sucrose gradient of about 5-50% at about 4°C or less.
  • the sedimented virus is collected at a suitable sucrose percentage (e.g., in the range of 5-50% sucrose), and sedimented again (after dilution with buffer) by ultracentrifugation at about 4°C or less.
  • the sedimented, purified virus is then suspended in buffer containing suitable preservatives. See, e.g., Portner et al, Virology 755:61-68 (1987); Takimoto et al, J. Virol. 66:1591-1600 (1992).
  • the purified virus can then be used for virosome preparation.
  • the protein be pure, in high concentration, biologically active, and/or have the transmembrane sequence removed. Removal ofthe transmembrane domains is preferred since aggregation ofthe transmembrane or hydrophobic domains can inhibit crystallization. These objectives are alternatively accomplished by foraiing virosomes, when the purified virus itself cannot be suitably cleaved to provide cleaved VAP for crystallization.
  • Virosomes comprise reconstituted viral lipid envelope or liposome, containing surface viral proteins. The surface proteins have lipophilic or hydrophobic portions in the viral envelope or liposome, as well as extra cellular portions projecting from the envelope or liposome.
  • the virosomes used in the present invention comprise a VAP such as hemagglutinin (HA), hemagglutinin neuraminidase (HN) or neuraminidase (NA), or other surface proteins that include, but are not limited to, F protein, sialidase, measles virus H protein, VSV G protein, gpl 20.
  • VAP such as hemagglutinin (HA), hemagglutinin neuraminidase (HN) or neuraminidase (NA), or other surface proteins that include, but are not limited to, F protein, sialidase, measles virus H protein, VSV G protein, gpl 20.
  • purified virus is added to a proportional volume of a saline buffer containing a suitable detergent (e.g. , non-ionic detergent) to solubilize the virus.
  • a suitable detergent e.g. , non-ionic detergent
  • the mixture is then incubated at about room temperature with shaking.
  • the preparation is then ultra-centrifuged at about 4°C or less to sediment the virus nucleocapsid and matrix proteins.
  • the supernatant containing at least one type of VAP is collected and the detergent removed.
  • the solution is then shaken at about room temperature or colder. Withdrawal of the detergent allows the virus membrane lipids and the virus envelope proteins to reform into a virosome as a lipid envelope containing the VAP extracellular portion projecting from the surface ofthe envelope.
  • the solution is collected and the procedure repeated to substantially remove the detergent.
  • the final solution contains the virosomes. See, e.g., Almeida et al, LANCET
  • the purified virosomes are optionally tested for biological activity (such as neuraminidase or sialidase activity) using known assays. See, e.g., Aymard-Henry et al, Bulletin ofthe World Health Organization, ⁇ 5.199-202 (1973); Thompson et al, J. Virol. 62:4653-4660 (1988); Takimoto et al, J. Virol. 66:1591-1600 (1992).
  • biological activity such as neuraminidase or sialidase activity
  • Proteolytic cleavage by a protease is used to remove soluble portions of a VAP, from the transmembrane portion, contained in either the virus or the virosome.
  • a proteolytic enzyme e.g., pronase
  • the preparation is ultra-centrifuged at about 4 °C or less. The cleaved VAP in the supernatant (as soluble protein) is collected and then optionally concentrated by further centrifugation.
  • the cleaved VAP is assayed for neuraminidase activity and for lack of aggregation, indicating that the transmembrane portion ofthe VAP has remained embedded in the virus envelope or virosome and is not part ofthe isolated protein. Removal ofthe hydrophobic membrane spanning region is preferred since aggregation ofthe hydrophobic regions can inhibit crystallization.
  • proteolytic treatment virosomes and cleaved VAP are separated by centrifugation, with the cleaved VAP remaining in the supernatant. For example, a modification of a procedure described previously can be used (Thompson et al, J. Virol. 62:4653-4660 (1988)).
  • the cleaved VAP fraction is preferably further concentrated by centrifugation through a filter, such as using a CENTRICON filter.
  • the results ofthe purification are optionally analyzed by polyacrylamide gel electrophoresis (PAGE) under reducing or non-reducing conditions. A single band is preferably obtained.
  • PAGE polyacrylamide gel electrophoresis
  • the analysis of the cleaved VAP be under non ⁇ reducing conditions to indicate whether the cleaved protein formed disulfide linked dimers.
  • the amino acid sequence can also be determined according to known methods, or otherwise obtained, as this sequence is important in determining the three dimensional structure ofthe cleaved protein (in combination with crystallographic analysis), as described herein, using molecular modeling techmques.
  • biological activity e.g., neuraminidase (or sialidase) activity for HN, or other activity ofthe VAP
  • biological activity is determined using equivalent amounts of both virion- or virosome- associated VAP and purified and cleaved protein. It is preferred that the biological activity exceed the activity of the virion- or virosome-associated protein. The preferred result indicates that the cleaved protein retains its native structure, which is important for determining the three-dimensional crystal structure of the biologically active molecule.
  • the purified and cleaved protein can be sequenced using known techniques. See, e.g. , Murti et al. , Proc. Natl. Acad. Sci. USA P ⁇ :1523-1525 (1993); Takimoto et al., J. Virol. 66:7597-7600 (1992), entirely incorporated herein by reference.
  • the hanging drop method is preferably used to crystallize the cleaved protein. See, e.g., Taylor et al., J. Mol. Biol. 226:1287-1290 (1992); Takimoto et ⁇ /., J. Virol. 66:7597-7600 (1992); CRYSTAL SCREEN, Hampton Research.
  • a mixture ofthe cleaved protein and precipitant can include the following: • pH (e.g., 4-9);
  • buffer type e.g., phosphate, sodium, or cacodylate acetates, imidazole, Tris HCl, sodium hepes
  • buffer concentration e.g., 10-200 mM
  • salt type e.g., calcium chloride, sodium citrate, magnesium chloride, ammonium acetate, ammonium sulfate, potassium phosphate, magnesium acetate, zinc acetate; calcium acetate
  • polymer type and concentration (e.g., polyethylene glycol (PEG) 1-50%, type 200- 10,000);
  • a non-limiting example of such crystalization conditions is the following: • purified cleaved protein (e.g., 5-30 mg/ml);
  • precipitant 2-60% Polyethylene glycol (PEG) 500-5000 buffered with 10-200 mM phosphate or acetate buffer and 50-300 mM of a precipitating salt (e.g., ammonium sulphate)); • at an overall pH of about 3.5-8.5.
  • a precipitating salt e.g., ammonium sulphate
  • the above mixtures are used and screened by varying at least one of pH, buffer type; buffer concentration, precipitating salt type or concentration, PEG type, PEG concentration, and cleaved protein concentration. Crystals ranging in size from 0.2-0.9 mm are formed in 1-14 days.
  • crystals diffract X-rays to at least 3.5 A resolution, such as 1.5 -3.5 A, or any range of value therein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0, with 3.0 ⁇ or less being preferred.
  • Crystals appear after 1-14 days and continue to grow on subsequent days. Some of the crystals are removed, washed, and assayed for biological activity, which activity is preferred for using in further characterizations. Other washed crystals are preferably run on a stained gel and those that migrate in the same position as the purified cleaved VAP are preferably used. From two to one hundred crystals are observed in one drop and crystal forms can occur, such as, but not limited to, bipyramidal, rhomboid, and cubic. Initial X-ray analyses indicate that such crystals diffract at moderately high to high resolution, such as 1.5-3.5 A or 2.2-2.7 A. When fewer crystals are produced in a drop, they can be much larger size, e.g., 0.4-0.9 mm.
  • antibody refers both to monoclonal antibodies which are a substantially homogeneous population and to polyclonal antibodies which are heterogeneous populations. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding antigen. Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and/or have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') fragments).
  • enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') fragments).
  • Antibodies can be generated against VAP produced recombinantly or isolated from cells and tissues where the VAP is present, as in virally infected cells. Antibodies can be generated against the entire VAP or, more preferably, antibodies are generated against peptide subfragments representing functional domains ofthe VAP required for its cell binding activity, e.g., the extracellular portion or a domain thereof. Antibodies for specifically inhibiting a VAP can be generated against peptide fragments unique to that protein. Alternatively, antibodies for generally inhibiting more than one member of a related class of VAPs can be generated against peptide fragments shared by the class of VAPs desired to be inhibited.
  • cDNA is generated from an envelope containing virus's RNA or virus-specific RNA from infected cells, or (in the case of DNA viruses) viral DNA is isolated, both from the virus and host cells containing the virus.
  • a suitable oligonucleotide, or set of oligonucleotides, which is complementary to a sequence encoding a VAP is identified and hybridized to the DNA or cDNA.
  • Single stranded oligonucleotide probes complementary to a unique portion of a VAP encoding sequence can be synthesized and labeled using known method steps. Such a probe can be used by known procedures (or as a basis for synthesizing PCR probes) for amplifying DNA encoding a VAP from an envelope containing virus. Such oligonucleotide probes can be at least about 10 nucleotides in length (such as 10-30, 30-100, 100-500, or any range or value therein), in order to be specific for a target VAP encoding nucleic acid. Such procedures are well-known in the art. See, e.g., Ausubel, infra, Sambrook, infra, and Kaufman, infra.
  • Culturing ofthe host and introduction of corresponding or complementary DNA or RNA into a vector and/or host cell can be performed by known methods. Any of a wide variety of vectors can be employed for this purpose. See, e.g., Ausubel, infra, ⁇ 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11.
  • a nucleic acid sequence encoding a VAP ofthe present invention can be recombined with vector DNA in accordance with conventional techniques, e.g. , as disclosed by Ausubel, infra, Kaufman, infra, or Sambrook, infra.
  • the vector is then incorporated into host cells (bacterial, yeast, insect or mammalian cells) using such vectors or viral vectors (e.g., vaccinia, a retrovirus, an adenovirus or a baculovirus), according to known techniques.
  • host cells bacterial, yeast, insect or mammalian cells
  • viral vectors e.g., vaccinia, a retrovirus, an adenovirus or a baculovirus
  • Host cells comprising a nucleic acid which encodes a VAP ofthe present invention can be grown under conditions that provide expression ofthe VAP in recoverable or commercially useful amounts. See, e.g., Ausubel, infra, at ⁇ 1 and 13; Palese, U.S. Patent No. 5,166,057, which are entirely inco ⁇ orated herein by reference.
  • NDV HN cDNA was cloning of NDV HN cDNA by RT-PCR (reverse transcriptase polymerase chain reaction). Briefly, viral mRNA was isolated from virus infected mammalian cells and was then reverse transcribed into cDNA. The cDNA was subjected to PCR amplification using gene-specific (NDV HN specific) primers (corresponding to the DNA sequence presented in Figure 5. The amplified cDNA, wliich encodes NDV HN gene, was ligated into vector plasmid and then the plasmid was introduced into E. coli.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • Virus (NDV) infected mammalian cells BHK cells were washed and suspended in a lysis buffer containing the nonionic detergent (Nonidet P-40). The intact nuclei were removed by a brief microfuge spin, and sodium dodecyl sulfate was added to the cytoplasm supernatant to denature protein. Protein was digested with protease and removed by extractions with phenol chloroform and chloroform. The cytoplasmic RNA which includes viral mRNA was recovered by ethanol precipitation. The isolated viral mRNA was used as a template to synthesize cDNA. First strand synthesis was driven by AMV reverse transcriptase and the oligo dT primer. Reverse transcriptases were derived from retroviruses such as avian myoblastosis virus (AMV) or Molony murine leukemia virus (MMLV), which use them to make DNA copies of their RNA genomes.
  • AMV avian myoblastosis virus
  • Oligonucleotides were used as primers for extension on RNA templates.
  • the DNA synthesized from the RNA template is complementary DNA (cDNA).
  • PCR was used to amplify a segment of the cDNA.
  • Two oligonucleotides were used as primers for a series of synthetic reactions that are catalyzed by a DNA polymerase (e.g., Taq DNA polymerase).
  • a DNA polymerase e.g., Taq DNA polymerase
  • These oligonucleotides are complementary to sequences that (1) lie on opposite strands of the template DNA and (2) flank the segment of DNA that is to be amplified.
  • These primers contain a potential restriction site at their 5' termini to facilitate cloning ofthe amplified double-stranded cDNA into an appropriate vector.
  • the major product of this reaction is a segment of double- stranded DNA whose termini are defined by the 5' ofthe oligonucleotide primers and whose length is defined by the distance between the primers.
  • the PCR product was cleaved with restriction enzyme which recognition sites were involved in the primers designed.
  • the NDV HN cDNA was then ligated into the plasmid vector pTFl (Takahashi et al, Genet. Anal Tech. Appl. 9:91-95 (1992)).
  • the NDV HN cDNA was subcloned into Hindlll and Kpnl sites of the pTFl vector.
  • the ligated DNA was introduced into Escherichia coli (E. coli). E. coli cells were transformed with the pTFl vector containing the NDV
  • HN cDNA using the calcium chloride precipitation method.
  • the transfected cells were grown in nonselective medium to allow synthesis of plasmid-encoded antibiotic resistance protein, then plated on antibiotic-containing medium to allow identification of plasmid containing colonies. Positive transformants were selected using ampicillin containing medium for the ampicillin resistance gene in the pTFl vector. Clones which included the plasmid pTFl with NDV HN cDNA insert were isolated, grown in the ampicillin-containing medium and, after adding glycerol to 50%, stored at -70 °C.
  • HN Hemagglutinin-Neuraminidase
  • Newcastle disease virus (Kansas strain) was diluted to 0.2 hemagglutination units (HA) in lOmM phosphate buffered saline (PBS pH7.4) containing gentamicin (0.5mg/ml: Bio Whittaker). Virus was inoculated into the allantoic cavity of 11 -day-old embryonated hen eggs (0.1 ml/egg). The eggs were incubated at 35 °C for two days and then chilled at 4°C overnight. The allantoic fluids were collected and centrifuged at 2,000 rpm for 30 min at 4°C in IEC CR-6000 centrifuge to remove red blood cells.
  • HA hemagglutination units
  • the virus in the supernatant was sedimented by ultracentrifugation at 30,000 rpm for 1 hr at 4°C. After the virus pellet was soaked in PBS overnight at 4°C, the pellet was resuspended by homogenization in a dounce homogenizer. The resuspended vims was purified by centrifugation in a 30-50% sucrose gradient (PBS) at 27,000 rpm for 2 hrs at 4°C. The vims which sedimented at approximately 40% sucrose was collected and sedimented again, after adding at least 1.5 vol of PBS, by ultracentrifugation at 35,000 rpm for 1 hr at 4°C. The sedimented purified vims was suspended in PBS containing 0.1% sodium azide.
  • PBS sucrose gradient
  • HN To prepare HN for crystallization, it is important that the HN be pure, in high concentration, biologically active, and have the transmembrane sequence removed. By forming virosomes these objectives were unexpectedly accomplished.
  • PBS PBS containing 2% Triton X-100 (Sigma) was added to solubilize the vims. The mixture was then incubated at room temperature for 1 hr with gentle shaking. The preparation was next centrifuged at 35,000 rpm for 2 hrs at 4°C to sediment the vims nucleocapsid and matrix proteins.
  • the supernatant containing HN and F proteins was collected and Bio-Beads (Bio-Rad) (1 gram/5 ml supernatant) added to remove the detergent.
  • the solution was gently shaken at room temperature for 1 hr. Withdrawal of the detergent allows the vims membrane lipids and the vims envelope proteins, HN and F, to reform into an envelope containing HN and F spikes projecting from the surface ofthe envelope.
  • the solution was collected by syringe with a 27G needle. The procedure was repeated twice more to remove the detergent completely.
  • the final solution contained the purified virosomes.
  • Proteolytic cleavage with a protease was used to remove HN from the virosome.
  • virosome solution 1.5mg/ml
  • pronase 0.5mg/ml in PBS
  • CACHEM pronase
  • the preparation was then centrifuged at 35,000 rpm for 1.5 hrs at 4°C.
  • the cleaved HN protein in the supernatant was concentrated by centrifugation through CENTRICON- 100 (AMICON) filter tubes. The concentrated HN was used for crystallization.
  • Figure 1 lane 3 shows the results of the purification, analyzed by polyacrylimide/gel electrophoresis (PAGE) under non-reducing conditions. A single band was obtained. Analysis of the HN under non-reducing conditions indicated that this HN ofthe Kansas strain of NDV did not form disulfide linked dimers. This strain is similar to the LaSota NDV strain which also does not show oligomeric HN under non-reducing PAGE analysis (Mirza et al, J. Biol. Chem. 265:21425- 21431 (1993)). The lack of cysteine in position 123 of the Kansas and LaSota strains (which is thought to be involved in disulfide bond formation) is likely responsible for the monomeric HN seen in the non-reducing gels.
  • PAGE polyacrylimide/gel electrophoresis
  • HN of this strain forms non-disulfide linked oligomers which are unstable under PAGE conditions. Additional characterization ofthe Kansas HN showed a protein migration pattem typical of NDV. We cloned and sequenced the Kansas HN gene (See Example 3) (Fig. 5) which showed a typical NDV HN sequence with up to 99% identity to HN from other NDV strains in the GenBank database. This sequence information is important in determining the three dimensional stmcture of HN from crystallographic analysis.
  • NDV HN Neuraminidase Activity of NDV HN Purified From Virosomes or on Virus Particles
  • HN Purified from HN Purified from of HN Protein ⁇ g
  • Virosomes A 549
  • HN Neuraminidase activity of equivalent amounts of HN, comparing the native viral activity with cleaved and purified HN.
  • the equivalent amounts of HN were incubated for 30 min at 37°C with 2.0 mg of N-acetylneuramin-lactose and then assayed for free sialic acid.
  • HN represents -25% of total virion protein.
  • the hanging drop method was used to crystallize the cleaved HN protein.
  • Crystals ranging in size from 0.2-0.7 mm were formed in 2-7 days (Fig. 2). Some of these crystals were removed, washed, and assayed for neuraminidase activity, which they were found to retain. Other washed crystals were run on a stained gel and found to migrate in the same position as the cleaved HN. As many as 40 crystals were observed in one drop and a number of different crystal forms were noted, including bipyramidal, rhomboid, and cubic. Initial X-ray analyses discussed in the next section indicates that the 0.2-0.25 mm crystal diffracts at moderately high resolution. Fewer crystals were also produced in a drop, but of much larger size, 0.4-0.6 mm.
  • the first crystals produced ( ⁇ 0.2-0.25 mm) were X-ray analyzed on a rotating Cu anode X- ray source operating at 40 kV and 100 mA.
  • Figure 3 shows the diffraction pattem from a single frame of several hundred collected. Crystals were stable for at least 20 hrs. Frozen crystals were used for longer X-ray exposures (48 hrs), the crystals being stable to the X-rays in the frozen state. To collect the maximum number of useful reflections, multiple frames were collected as the crystal was rotated in the X-ray beam for 48 hrs. In this analysis, crystals diffracted to a resolution of 3.5 A (Fig. 4, edge). To increase the resolution further, slightly larger crystals (0.25 mm) were analyzed in a synchrotron high energy X- ray source. Using frozen crystals, X-ray diffraction data was collected every 6 minutes over a 24-hr period. A single frame is shown in Figure 5. The crystals diffracted to a relatively high resolution of 2.6 A.
  • Example 3 Cloning and Sequencing of Nucleic Acid Encoding a Paramyxovirus HN Protein
  • HN gene of NDV was cloned by polymerase chain reaction (PCR) method using RNA extracted from vims infected BHK cells.
  • First strand cDNA was synthesized by Moloney murine leukemia vims reverse transcriptase (Promega) using primer designed from consensus sequence found in NDV strains obtained from NIH GenBank. The synthesized cDNA was amplified by PCR using similarly designed primers.
  • NDV HNcDNA containing full coding region was subcloned into plasmid pTFl (Takahashi et al, 1992, Bousse et al, 1994) at Hindlll and Kpnl sites.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox, P.L.L.C.
  • GCA AAA AAT ACA TGG CGC TTG ATA TTC CGG ATT GCA ATC TTA CTC TTA 96 Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Leu Leu 20 25 30

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Abstract

The invention provides purification methods and crystallized viral attachment protein (VAP) of virosomes derived from a virus, wherein (a) the crystallized VAP is suitable for x-ray crystallography analysis; (b) x-ray analysis provides diffraction patterns of sufficient resolution to determine the three-dimensional structure of the VAP; (c) the crystallized VAP is in biologically active form, as well as a specific crystallized VAP, hemagglutinin neuraminidase (HN) from a strain of Paramyxovirus, including nucleic acid, vectors and host cells having nucleotide sequences encoding the HN.

Description

Virus Protein Purification from Virosomes
Background ofthe Invention
Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development
Part ofthe work performed during development of this invention utilized U.S. Government funds as NIH Grant No. 5R01 AH 1949-20. The U.S. Government has certain rights in this invention.
Field ofthe Invention
The present invention generally pertains to the field of viral protein crystallization. The present invention specifically pertains to crystallization methods and crystallized viral attachment proteins (VAPs), obtained from virosomes. The crystallized VAP is biologically active.
Crystallized VAP, nucleic acid, vectors and host cells of a strain of paramyxovirus are also provided.
Related Art
VAP and Infection
Many infectious viruses contain an inner virion core having nucleic acid and a lipid envelope which holds the transmembrane (hydrophobic) domains of the envelope proteins. Some of the envelope proteins are viral attachment proteins (VAPs) that contain extracellular domains. Viruses infect a target cell by association ofthe virus' VAP with the target cell's viral receptor.
The extracellular domain ofthe VAP binds the target cell receptor and the transmembrane domain anchors the VAP to the viral envelope. (White et al, Quant. Rev. Biophys. 16: 151-195 (1983)). After association of the VAP with the cell's viral receptor, the virion core enters the cytoplasm ofthe bound cell and the viral replication process is initiated. In some cases, viruses that are bound to target cell receptors can enter the cells by receptor mediated endocytosis. Infectious Envelope-Containing Viruses
Examples of infectious envelope-containing viruses include, but are not limited to, togaviruses (yellow fever, RSSE and rubella); retroviruses (leukemia, sarcomas); orthomyxoviruses (influenza A, B, C); paramyxoviruses (mumps, measles, paramfluenza, Newcastle disease); rhabdoviruses (rabies); hepatitis virus; herpes viruses (herpes simplex, varicella zoster, cytomegalovirus, Epstein-Barr); and poxviruses (varcola, vaccinia, Molluscum Contagiosum). See, e.g., Lycke andNorrby, eds. Textbook of Medical Virology, Chs. 1-4 and 7-9, Butterworths, London (1983).
Paramyxoviruses
Paramyxoviruses are one ofthree genera ofthe family Paramyxoviridae, which includes enveloped, negative-stranded RNA viruses. Paramyxoviruses utilize hemagglutinin neuraminidase (HN) as the target VAP (Fraenkel-Conrat and Wagner, eds., Comprehensive Virology, Vol. 4,
Plenum Publishing Corp., New York ( 1975), pp. 99- 178). Important examples of paramyxoviruses include mumps, measles, parainfluenza virus (PIV), Sendai virus (SV) and Newcastle disease virus (NDV).
Epidemiologically, particular strains of paramyxovirus are responsible for annual epidemics in humans. Reinfection by the same or similar strains in subsequent years is common, although less severe (Kass, ed., Studies in Infectious Diseases Research, The University of Chicago Press, Chicago (1975), pp. 51-64).
While some immunity develops through natural infection by most strains of paramyxoviruses, this immunity is generally not sufficient to provide complete protection. Although a few vaccines have been developed for some paramyxoviruses, these vaccines have limited effectiveness in the short term, and are generally ineffective in the long term (Choppin and Scheid, Rev. Infect. Dis. 2:40-61 (1980); Norrby et al., J. Infect. Dis. 132:262-269 (1975)). Structural Organization ofthe Paramyxoviruses
Electron micrographs have demonstrated that all paramyxovirus species have the same basic morphology. This morphology includes highly pleomorphic particles that are enclosed by a lipid envelope acquired during maturation. Maturation usually occurs by virus budding from the plasma membrane ofthe host cell.
The viral membrane of a paramyxovirus contains two virus-specified glycoproteins, HN and F. HN and F are found in all strains of paramyxoviruses. HN and F are attached to the viral membrane by short N-terminal and C-teιτninal transmembrane peptide sequences, respectively. The nucleotide sequence of HN genes of several paramyxoviruses has been determined. See, e.g., Gorman etal, Virology 775:211-221 (1990); Merson etal, Virology 167:91-105 (1988)); Blumberg et al., Cell 41:269-218 (1985); Paterson et al, Proc. Natl. Acad. Sci. USA 81:6106-6110 (1984); Hiebert etα/., J Virol. 53:1-6 (1985); Hsu and Choppin, Proc. Natl. Acad. Sci. USA 81:1132-1136 (1984); Thompson et al, J. Virol. (52:4653-4660 (1988).
Biological Activities ofthe Paramyxovirus
Paramyxovirus infection is initiated by the action of the two glycoproteins, HN and F (Kingsbury, supra, pp. 347-382; Fields, ed., Virology, Raven Press, New York (1985), pp. 1241- 1253). HN protein from different paramyxovirus strains exhibit the same biological activities.
These activities include hemagglutination (HA), cell binding, neuraminidase (NA) activity, and fusion promoting activities (Fraenkel-Conrat, supra, pp. 99-178 and 293-407; Kessler et al, J. Gen. Virol. 37:541-556 (1977); Scheid and Choppin, Virology 62:125-133 (1974); Ebata et ai, Virology 755:437-441 (1991); Morrison etα/., J. Virol. (55:813-822 (1991); Hu et α/., J Virol. 66: 1528-1534 (1992); Tanabayashi et al., Virology 757:801-804 (1992); Horvath et al, J. Virol. r5<5:4564-4569
(1992)).
Hemagglutination activity is the capacity of a virus to absorb to erythrocytes and, as a result, cause the erythrocytes to aggregate (agglutinate). For example, in the paramyxoviruses a protein projecting from the virus membrane surface (HN) mediates the attachment to a sialic acid glyco- conjugate receptors on the erythrocyte surface. The hemagglutination reaction (HA) is an example of a relatively simple, quick, convenient and semi-quantitative way of detecting, identifying, titrating viruses, detecting viral antibody and studying virus attachment. Cell-binding activity is the capacity ofa virus to attach to a variety of infectible host cells. The HN protein of paramyxoviruses mediates the attachments to host cells, via a sialic acid-containing glyco-conjugate receptor. Neuraminidase activity is the enzyme catalyzed cleavage of the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue. The HN protein of paramyxoviruses possess neuraminidase activity. Fusion promoting activity is the capacity of paramyxovirus VAPs (e.g., HN, H) to provide an essential function that allows the fusion (F) protein to directly mediate virus host-cell and cell-to-cell membrane fusion. (Fraenkel-Conrat, supra, pp. 99-178 and 293-407; Kessler et al, J. Gen. Virol. 37:541-556 (1977); Scheid and Choppin, Virology 62:125-133 (1974); Ebata etα/., Virology 183:431-441 (1991); Morrison etal, J. Virol. 65:813-822 (1991); Hu etα/., J. Virol. 66:1528-1534 (1992); Tanabayashi et al, Virology 757:801-804 (1992); Horvath et al, J. Virol. 66:4564-4569 (1992)).
Structural Separation ofHN Biological Activities
The structural separation ofthe biological activities ofthe HN of paramyxoviruses has been partially demonstrated in such species as NDV, SV, PIV-1, and PIV-3. Attachment, neuraminidase and fusion promoting functions of both the NDV HN and the S V HN have been dissociated by binding with different MAbs. Additionally, specific mutations of HN cDNA resulted in expressed HN protein having one particular function inhibited, but not others. See, e.g., Bousse et al, Virology 204:506-514 (1994); Bishop & Compans, eds., Nonsegmented Negative Strand Viruses, Academic Press, Orlando, Florida (1984), pp. 345-350; Portner et al, Virology 755:61-68 (1987); Iorio andBratt,J. 7/w7WMr2o/. 133:2115-2119 (1984); Iorio etα/.,J. Gen. Virol. 75:1167-1176 (1992); Sergeletα/., Virology 193:111-126 (1993); Sergei etal, ϊro/ogy 7P6:831-834 (1993). Suchresults suggest that the active sites on HN for these biological activities are separate.
HN cell-binding, neuraminidase, and fusion promoting activities, essential for virus infection and spreading, are conserved among all or most strains of paramyxoviruses, as reflected in the high degree of sequence identity among these proteins. Therefore, the determined three-dimensional structure of an HN from a strain of paramyxovirus is useful for rational design of inhibitors to treat infections of many or most paramyxoviruses and may be applicable to other members of Paramyxoviridae family. By analogy, the crystal structures of neuramimdases, from influenza virus, Salmonella tryphimurium, and Vibrio cholerae show similar three-dimensional structures (Crennell et al, Structure 2:535-544 (1994); Crennell et al, Proc. Natl. Acad. Sci. USA 0:9852-9856 (1993)).
Summary ofthe Invention
The present invention provides methods of purifying and crystallizing a viral attachment protein (VAP) from an envelope containing virus. The present invention also provides crystallized VAP which is soluble and biologically active.
The present invention also provides antibodies specific for the VAP and host cells that produce the antibody.
The present invention further provides nucleic acid molecules encoding the VAP, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the molecule.
The present invention also provides a crystallized HN protein from a strain ofa species of a paramyxovirus.
The present invention also provides antibodies specific for the HN and host cells that produce the antibody.
The present invention further provides nucleic acid molecules encoding the HN, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the nucleic acid.
The present invention also provides a crystallized HN protein from the Kansas strain of a species of a paramyxovirus: Newcastle disease virus (NDV). This HN crystallized protein is suitable for x-ray diffraction analysis. The x-ray diffraction patterns obtained by this analysis provide coordinates of moderately high to high resolution. These coordinates are useful for three dimensional modeling of the HN protein. The three dimensional modeling programs use these coordinates and the amino acid sequence to generate secondary, tertiary and quaternary structures ofthe Kansas NDV HN. The present invention also provides antibodies specific for the Kansas NDV HN and antibody expressing host cells.
The present invention further provides nucleic acid molecules encoding the Kansas NDV HN, as well as nucleic acid probes specific for portions ofthe nucleic acid molecule. Also provided are vectors and host cells comprising the nucleic acid.
Other objects ofthe invention will be apparent to one of ordinary skill in the art from the following detailed description and examples relating to the present invention.
Brief Description ofthe Figures
Figure ! Results are shown from the non-reduced SDS-polyacrylamide gel electrophoresis of protease-cleaved NH protein from NDV. Lane 1 contains 20 μg of purified virus. Lane 2 contains an envelope fraction containing 5 μg of uncleaved HN. Lane 3 contains 10 μg of cleaved HN. The samples in the SDS loading buffer lacked reducing agent (1% B-mercaptoethanol) and were boiled for 2 minutes before loading. HN under reducing conditions migrated in a similar position. No disulfide linked oligomers were thus evident.
Figure 2. Results are shown from the crystallization of purified cleaved HN using the hanging drop-vapor diffusion method. 1.0 μl of cleaved HN solution (10 mg/ml) was mixed with an equal amount of precipitant and left to equilibrate over a reservoir ofthe same composition as the precipitant and deionized water. The reservoir solution was composed of 0.5 ml 20% (w/v) PEG
4000, 0.16 M ammonium sulfate, buffered with sodium acetate, pH 4.6.
Figure 3. A diffraction pattern is presented from a crystal of Kansas strain NDV cleaved HN using an X-ray source. The resolution was 3.5 A at the edge ofthe pattern.
Figure 4. A diffraction pattern is presented from a crystal of cleaved HN using a more powerful X-ray beam than in Figure 3, produced in synchrotron storage rings. The resolution ofthe pattern was 2.6 A at the edge. Data was collected using a crystal frozen at -175 °C.
Figure 5. The nucleotide sequence ofthe Kansas strain of NDV is presented. Figure 6. The deduced amino acid sequence of an HN ofthe Kansas strain of NDV is presented.
Detailed Description ofthe Preferred Embodiments
The present invention overcomes one or more deficiencies ofthe related background art, by providing methods for crystallizing a viral attachment protein (VAP) from virosomes, where the crystals diffract x-rays with high resolution of 1.5-3.9A, such as 2.4-27A.
The present invention thus includes methods of purifying and crystallizing a VAP from virosomes derived from a virus. The present invention also provides crystallized VAP by these methods which is soluble and biologically active.
The present invention, in a non-limiting example, provides methods of purifying and crystallizing hemagglutinin neuraminidase (HN) from a strain of a paramyxovirus using virosomes. The present invention also provides crystallized HN by these methods which is soluble and biologically active.
The present invention also provides biologically active VAPs. A non-limiting example is an HN from the Kansas strain of a species ofa paramyxovirus, the Newcastle disease virus (NDV). The VAP is also provided as a crystallized protein.
Overview of VAP Purification and Crystallization Methods
In general, a VAP from a virus is isolated in soluble form (e.g., lacking the transmembrane domains) by cleavage employing a protease applied to purified viruses or virosomes, as described herein. The resulting cleaved VAP is in sufficient purity and concentration (e.g., a monomer or dimer) for crystallization. The cleaved VAP is then isolated and assayed for biological activity and for lack of aggregation (which interferes with crystallization). The purified and cleaved VAP preferably runs as a single band under reducing or nonreducing polyacrylamide gel electrophoresis (PAGE) (nonreducing is used to evaluate the presence of cysteine bridges).
The purified cleaved VAP is preferably crystallized using the hanging drop method under varying conditions of at least one ofthe following: pH, buffer type, buffer concentration, salt type, -o-
polymer type, polymer concentration, other precipitating agents and concentration of purified and cleaved VAP. See, e.g., the methods provided in a commercial kit, such as CRYSTAL SCREEN (Hampton Research, Riverside, CA). The crystallized protein is also tested for neuraminidase or cell binding biological activity and differently sized and shaped crystals are further tested for suitability for X-ray diffraction. Generally, larger crystals provide better crystallography than smaller crystals, and thicker crystals provide better crystallography than thinner crystals.
Virus Culture and Isolation Methods
To prepare isolated virus for purification of VAPs from virosomes, a strain of virus is diluted in a buffer solution at about neutral pH. The diluted virus solution can also be inoculated into the allantoic cavity of embryonated hen eggs for amplification. Tissue culture of a virus strain, or recombinant expression ofthe VAP can alternatively be used according to known method steps.
When allantoic culture is used, infected, embroynated eggs are incubated for several days and then chilled at about 4°C or less overnight. The allantoic fluids are collected and centrifuged at about 4°C or less to remove red blood cells. The virus in the supernatant is sedimented by ultracentrifugation at about 4°C or less. After the virus pellet is soaked in buffer solution overnight at about 4°C or less, the pellet is resuspended, e.g., by homogenization.
The resuspended virus is optionally further purified by centrifugation in a sucrose gradient of about 5-50% at about 4°C or less. The sedimented virus is collected at a suitable sucrose percentage (e.g., in the range of 5-50% sucrose), and sedimented again (after dilution with buffer) by ultracentrifugation at about 4°C or less. The sedimented, purified virus is then suspended in buffer containing suitable preservatives. See, e.g., Portner et al, Virology 755:61-68 (1987); Takimoto et al, J. Virol. 66:1591-1600 (1992). The purified virus can then be used for virosome preparation.
Methods for Preparation of Purified Virosomes
To prepare HN or other VAP for crystallization, it is preferred that the protein be pure, in high concentration, biologically active, and/or have the transmembrane sequence removed. Removal ofthe transmembrane domains is preferred since aggregation ofthe transmembrane or hydrophobic domains can inhibit crystallization. These objectives are alternatively accomplished by foraiing virosomes, when the purified virus itself cannot be suitably cleaved to provide cleaved VAP for crystallization. Virosomes comprise reconstituted viral lipid envelope or liposome, containing surface viral proteins. The surface proteins have lipophilic or hydrophobic portions in the viral envelope or liposome, as well as extra cellular portions projecting from the envelope or liposome. The virosomes used in the present invention comprise a VAP such as hemagglutinin (HA), hemagglutinin neuraminidase (HN) or neuraminidase (NA), or other surface proteins that include, but are not limited to, F protein, sialidase, measles virus H protein, VSV G protein, gpl 20.
To form virosomes, purified virus is added to a proportional volume of a saline buffer containing a suitable detergent (e.g. , non-ionic detergent) to solubilize the virus. The mixture is then incubated at about room temperature with shaking. The preparation is then ultra-centrifuged at about 4°C or less to sediment the virus nucleocapsid and matrix proteins. The supernatant containing at least one type of VAP is collected and the detergent removed. The solution is then shaken at about room temperature or colder. Withdrawal of the detergent allows the virus membrane lipids and the virus envelope proteins to reform into a virosome as a lipid envelope containing the VAP extracellular portion projecting from the surface ofthe envelope. The solution is collected and the procedure repeated to substantially remove the detergent. The final solution contains the virosomes. See, e.g., Almeida et al, LANCET, Nov. 8, 1975, 899-901.
The purified virosomes are optionally tested for biological activity (such as neuraminidase or sialidase activity) using known assays. See, e.g., Aymard-Henry et al, Bulletin ofthe World Health Organization, ¥5.199-202 (1973); Thompson et al, J. Virol. 62:4653-4660 (1988); Takimoto et al, J. Virol. 66:1591-1600 (1992).
Viral Protein Purification Methods
Proteolytic cleavage by a protease is used to remove soluble portions of a VAP, from the transmembrane portion, contained in either the virus or the virosome. To a virosome solution is added a proteolytic enzyme (e.g., pronase), and the mixture incubated at about room temperature overnight. To remove the virosomes, the preparation is ultra-centrifuged at about 4 °C or less. The cleaved VAP in the supernatant (as soluble protein) is collected and then optionally concentrated by further centrifugation.
The cleaved VAP is assayed for neuraminidase activity and for lack of aggregation, indicating that the transmembrane portion ofthe VAP has remained embedded in the virus envelope or virosome and is not part ofthe isolated protein. Removal ofthe hydrophobic membrane spanning region is preferred since aggregation ofthe hydrophobic regions can inhibit crystallization. After proteolytic treatment, virosomes and cleaved VAP are separated by centrifugation, with the cleaved VAP remaining in the supernatant. For example, a modification of a procedure described previously can be used (Thompson et al, J. Virol. 62:4653-4660 (1988)). The cleaved VAP fraction is preferably further concentrated by centrifugation through a filter, such as using a CENTRICON filter.
Purified Viral Proteins
The results ofthe purification are optionally analyzed by polyacrylamide gel electrophoresis (PAGE) under reducing or non-reducing conditions. A single band is preferably obtained. With disulfide-containing VAPs, it is preferred that the analysis of the cleaved VAP be under non¬ reducing conditions to indicate whether the cleaved protein formed disulfide linked dimers. The amino acid sequence can also be determined according to known methods, or otherwise obtained, as this sequence is important in determining the three dimensional structure ofthe cleaved protein (in combination with crystallographic analysis), as described herein, using molecular modeling techmques.
Before crystallization, biological activity (e.g., neuraminidase (or sialidase) activity for HN, or other activity ofthe VAP) is determined using equivalent amounts of both virion- or virosome- associated VAP and purified and cleaved protein. It is preferred that the biological activity exceed the activity of the virion- or virosome-associated protein. The preferred result indicates that the cleaved protein retains its native structure, which is important for determining the three-dimensional crystal structure of the biologically active molecule. To identify the protease cleavage site, the purified and cleaved protein can be sequenced using known techniques. See, e.g. , Murti et al. , Proc. Natl. Acad. Sci. USA Pø:1523-1525 (1993); Takimoto et al., J. Virol. 66:7597-7600 (1992), entirely incorporated herein by reference.
Viral Protein Crystallization Methods
The hanging drop method is preferably used to crystallize the cleaved protein. See, e.g., Taylor et al., J. Mol. Biol. 226:1287-1290 (1992); Takimoto et α/., J. Virol. 66:7597-7600 (1992); CRYSTAL SCREEN, Hampton Research.
A mixture ofthe cleaved protein and precipitant can include the following: • pH (e.g., 4-9);
• buffer type (e.g., phosphate, sodium, or cacodylate acetates, imidazole, Tris HCl, sodium hepes);
• buffer concentration (e.g., 10-200 mM);
• salt type (e.g., calcium chloride, sodium citrate, magnesium chloride, ammonium acetate, ammonium sulfate, potassium phosphate, magnesium acetate, zinc acetate; calcium acetate)
• polymer type and concentration: (e.g., polyethylene glycol (PEG) 1-50%, type 200- 10,000);
• other precipitating agents (salts: K, Na, tartrate, NH4SO4, NaAc, LiSO4, NaFormate, NaCitrate, MgFormate, NaPO4, KPO4 NH4PO4; organics: 2-propanol; non-volatile:
2-methyl-2,4-pentanediol); and
• concentration of purified cleaved VAP (e. g. , 5.0- 100 mg/ml) . See, e.g., CRYSTAL SCREEN, Hampton Research.
A non-limiting example of such crystalization conditions is the following: • purified cleaved protein (e.g., 5-30 mg/ml);
• H2O;
• precipitant 2-60% Polyethylene glycol (PEG) 500-5000 buffered with 10-200 mM phosphate or acetate buffer and 50-300 mM of a precipitating salt (e.g., ammonium sulphate)); • at an overall pH of about 3.5-8.5. The above mixtures are used and screened by varying at least one of pH, buffer type; buffer concentration, precipitating salt type or concentration, PEG type, PEG concentration, and cleaved protein concentration. Crystals ranging in size from 0.2-0.9 mm are formed in 1-14 days. These crystals diffract X-rays to at least 3.5 A resolution, such as 1.5 -3.5 A, or any range of value therein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0, with 3.0 Λ or less being preferred.
Viral Protein Crystals
Crystals appear after 1-14 days and continue to grow on subsequent days. Some of the crystals are removed, washed, and assayed for biological activity, which activity is preferred for using in further characterizations. Other washed crystals are preferably run on a stained gel and those that migrate in the same position as the purified cleaved VAP are preferably used. From two to one hundred crystals are observed in one drop and crystal forms can occur, such as, but not limited to, bipyramidal, rhomboid, and cubic. Initial X-ray analyses indicate that such crystals diffract at moderately high to high resolution, such as 1.5-3.5 A or 2.2-2.7 A. When fewer crystals are produced in a drop, they can be much larger size, e.g., 0.4-0.9 mm.
Production and Use of Antibodies Specific to a VAP
The term "antibody", as used herein, refers both to monoclonal antibodies which are a substantially homogeneous population and to polyclonal antibodies which are heterogeneous populations. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The term "antibody", as used herein, is also meant to include both intact molecules as well as fragments thereof, such as Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and/or have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med. 2^:316-325 (1983)). Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') fragments). See, generally, Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al, eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1987, 1992, 1993, 1994); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988); Colligan et al, eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), the contents of which references are entirely incorporated herein by reference.
Both monoclonal and polyclonal antibodies to a VAP, in crystalline or non-crystalline form, can be made according to methods well known in the art (see, e.g., Harlow, supra; Colligan, supra; Ausubel, supra, at §§11.4.2-11.13.4). Antibodies can be generated against VAP produced recombinantly or isolated from cells and tissues where the VAP is present, as in virally infected cells. Antibodies can be generated against the entire VAP or, more preferably, antibodies are generated against peptide subfragments representing functional domains ofthe VAP required for its cell binding activity, e.g., the extracellular portion or a domain thereof. Antibodies for specifically inhibiting a VAP can be generated against peptide fragments unique to that protein. Alternatively, antibodies for generally inhibiting more than one member of a related class of VAPs can be generated against peptide fragments shared by the class of VAPs desired to be inhibited.
Cloning and Expression of Nucleic Acid Encoding a VAP
Known method steps for synthesizing oligonucleotides probes useful for cloning DNA encoding a VAP, such as an HN or HA, based on the teaching and guidance presented herein, are disclosed by, for example, in Ausubel, infra; Sambrook et al, Molecular Cloning: A Laboratory
Manual, Second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); and
Kaufman et al, eds. Handbook of Molecular and Cellular Methods in Biology and Medicine, CRC
Press, Inc., Boca Raton (1995), which references are entirely incorporated herein by reference. cDNA is generated from an envelope containing virus's RNA or virus-specific RNA from infected cells, or (in the case of DNA viruses) viral DNA is isolated, both from the virus and host cells containing the virus. A suitable oligonucleotide, or set of oligonucleotides, which is complementary to a sequence encoding a VAP is identified and hybridized to the DNA or cDNA.
Single stranded oligonucleotide probes complementary to a unique portion of a VAP encoding sequence can be synthesized and labeled using known method steps. Such a probe can be used by known procedures (or as a basis for synthesizing PCR probes) for amplifying DNA encoding a VAP from an envelope containing virus. Such oligonucleotide probes can be at least about 10 nucleotides in length (such as 10-30, 30-100, 100-500, or any range or value therein), in order to be specific for a target VAP encoding nucleic acid. Such procedures are well-known in the art. See, e.g., Ausubel, infra, Sambrook, infra, and Kaufman, infra.
Culturing ofthe host and introduction of corresponding or complementary DNA or RNA into a vector and/or host cell can be performed by known methods. Any of a wide variety of vectors can be employed for this purpose. See, e.g., Ausubel, infra, §§ 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11. A nucleic acid sequence encoding a VAP ofthe present invention can be recombined with vector DNA in accordance with conventional techniques, e.g. , as disclosed by Ausubel, infra, Kaufman, infra, or Sambrook, infra. The vector is then incorporated into host cells (bacterial, yeast, insect or mammalian cells) using such vectors or viral vectors (e.g., vaccinia, a retrovirus, an adenovirus or a baculovirus), according to known techniques.
Host cells comprising a nucleic acid which encodes a VAP ofthe present invention can be grown under conditions that provide expression ofthe VAP in recoverable or commercially useful amounts. See, e.g., Ausubel, infra, at §§ 1 and 13; Palese, U.S. Patent No. 5,166,057, which are entirely incoφorated herein by reference.
Cloning ofNDVHNcDNA
As a non-limiting example, cloning of NDV HN cDNA was performed by RT-PCR (reverse transcriptase polymerase chain reaction). Briefly, viral mRNA was isolated from virus infected mammalian cells and was then reverse transcribed into cDNA. The cDNA was subjected to PCR amplification using gene-specific (NDV HN specific) primers (corresponding to the DNA sequence presented in Figure 5. The amplified cDNA, wliich encodes NDV HN gene, was ligated into vector plasmid and then the plasmid was introduced into E. coli.
Virus (NDV) infected mammalian cells (BHK cells) were washed and suspended in a lysis buffer containing the nonionic detergent (Nonidet P-40). The intact nuclei were removed by a brief microfuge spin, and sodium dodecyl sulfate was added to the cytoplasm supernatant to denature protein. Protein was digested with protease and removed by extractions with phenol chloroform and chloroform. The cytoplasmic RNA which includes viral mRNA was recovered by ethanol precipitation. The isolated viral mRNA was used as a template to synthesize cDNA. First strand synthesis was driven by AMV reverse transcriptase and the oligo dT primer. Reverse transcriptases were derived from retroviruses such as avian myoblastosis virus (AMV) or Molony murine leukemia virus (MMLV), which use them to make DNA copies of their RNA genomes.
Oligonucleotides were used as primers for extension on RNA templates. The DNA synthesized from the RNA template is complementary DNA (cDNA). PCR was used to amplify a segment of the cDNA. Two oligonucleotides were used as primers for a series of synthetic reactions that are catalyzed by a DNA polymerase (e.g., Taq DNA polymerase). These oligonucleotides are complementary to sequences that (1) lie on opposite strands of the template DNA and (2) flank the segment of DNA that is to be amplified. These primers contain a potential restriction site at their 5' termini to facilitate cloning ofthe amplified double-stranded cDNA into an appropriate vector. The major product of this reaction is a segment of double- stranded DNA whose termini are defined by the 5' ofthe oligonucleotide primers and whose length is defined by the distance between the primers. The PCR product was cleaved with restriction enzyme which recognition sites were involved in the primers designed.
The NDV HN cDNA was then ligated into the plasmid vector pTFl (Takahashi et al, Genet. Anal Tech. Appl. 9:91-95 (1992)). The NDV HN cDNA was subcloned into Hindlll and Kpnl sites of the pTFl vector. After litigation of vector DNA, the ligated DNA was introduced into Escherichia coli (E. coli). E. coli cells were transformed with the pTFl vector containing the NDV
HN cDNA using the calcium chloride precipitation method. The transfected cells were grown in nonselective medium to allow synthesis of plasmid-encoded antibiotic resistance protein, then plated on antibiotic-containing medium to allow identification of plasmid containing colonies. Positive transformants were selected using ampicillin containing medium for the ampicillin resistance gene in the pTFl vector. Clones which included the plasmid pTFl with NDV HN cDNA insert were isolated, grown in the ampicillin-containing medium and, after adding glycerol to 50%, stored at -70 °C. Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration, and is not intended to be limiting ofthe present invention.
Example 1: Preparation, Purification and Crystallization of
Hemagglutinin-Neuraminidase (HN) from a Paramyxovirus Using Virosomes
Propagation and purification of virus
Newcastle disease virus (Kansas strain) was diluted to 0.2 hemagglutination units (HA) in lOmM phosphate buffered saline (PBS pH7.4) containing gentamicin (0.5mg/ml: Bio Whittaker). Virus was inoculated into the allantoic cavity of 11 -day-old embryonated hen eggs (0.1 ml/egg). The eggs were incubated at 35 °C for two days and then chilled at 4°C overnight. The allantoic fluids were collected and centrifuged at 2,000 rpm for 30 min at 4°C in IEC CR-6000 centrifuge to remove red blood cells. The virus in the supernatant was sedimented by ultracentrifugation at 30,000 rpm for 1 hr at 4°C. After the virus pellet was soaked in PBS overnight at 4°C, the pellet was resuspended by homogenization in a dounce homogenizer. The resuspended vims was purified by centrifugation in a 30-50% sucrose gradient (PBS) at 27,000 rpm for 2 hrs at 4°C. The vims which sedimented at approximately 40% sucrose was collected and sedimented again, after adding at least 1.5 vol of PBS, by ultracentrifugation at 35,000 rpm for 1 hr at 4°C. The sedimented purified vims was suspended in PBS containing 0.1% sodium azide.
Forming virosomes by detergent withdrawal method
To prepare HN for crystallization, it is important that the HN be pure, in high concentration, biologically active, and have the transmembrane sequence removed. By forming virosomes these objectives were unexpectedly accomplished. To purified vims (20mg/ml), an equal volume of PBS containing 2% Triton X-100 (Sigma) was added to solubilize the vims. The mixture was then incubated at room temperature for 1 hr with gentle shaking. The preparation was next centrifuged at 35,000 rpm for 2 hrs at 4°C to sediment the vims nucleocapsid and matrix proteins. The supernatant containing HN and F proteins was collected and Bio-Beads (Bio-Rad) (1 gram/5 ml supernatant) added to remove the detergent. The solution was gently shaken at room temperature for 1 hr. Withdrawal of the detergent allows the vims membrane lipids and the vims envelope proteins, HN and F, to reform into an envelope containing HN and F spikes projecting from the surface ofthe envelope. The solution was collected by syringe with a 27G needle. The procedure was repeated twice more to remove the detergent completely. The final solution contained the purified virosomes.
Isolation and purification of HN protein
Proteolytic cleavage with a protease was used to remove HN from the virosome. To 4 volumes of virosome solution (1.5mg/ml), 1 volume of pronase (0.5mg/ml in PBS)(CALBIOCHEM) was added and the mixture incubated at room temperature ovemight. To collect the virosomes, the preparation was then centrifuged at 35,000 rpm for 1.5 hrs at 4°C. The cleaved HN protein in the supernatant was concentrated by centrifugation through CENTRICON- 100 (AMICON) filter tubes. The concentrated HN was used for crystallization.
Figure 1, lane 3, shows the results of the purification, analyzed by polyacrylimide/gel electrophoresis (PAGE) under non-reducing conditions. A single band was obtained. Analysis of the HN under non-reducing conditions indicated that this HN ofthe Kansas strain of NDV did not form disulfide linked dimers. This strain is similar to the LaSota NDV strain which also does not show oligomeric HN under non-reducing PAGE analysis (Mirza et al, J. Biol. Chem. 265:21425- 21431 (1993)). The lack of cysteine in position 123 of the Kansas and LaSota strains (which is thought to be involved in disulfide bond formation) is likely responsible for the monomeric HN seen in the non-reducing gels. HN of this strain forms non-disulfide linked oligomers which are unstable under PAGE conditions. Additional characterization ofthe Kansas HN showed a protein migration pattem typical of NDV. We cloned and sequenced the Kansas HN gene (See Example 3) (Fig. 5) which showed a typical NDV HN sequence with up to 99% identity to HN from other NDV strains in the GenBank database. This sequence information is important in determining the three dimensional stmcture of HN from crystallographic analysis. Before crystallization was undertaken, we measured the neuraminidase activity in equivalent amounts of virion associated HN and purified cleaved HN and found that cleaved HN activity was equivalent with the activity ofthe virion-associated HN (Table 3). This indicated that the cleaved HN retained its native structure, which is important for determining the three-dimensional crystal stmcture of a biologically active molecule. To identify the protease cleavage site, the purified cleaved HN was sequenced using automated Edman degradation. An exact match of 15 amino acids at the newly created amino terminus ofthe cleaved HN identified the cleavage site at Gly 124 ("f " in Figure 6).
Table 3
Neuraminidase Activity of NDV HN Purified From Virosomes or on Virus Particles
Activity of Activity
Equivalent Amount HN Purified from HN Purified from of HN Protein (μg) Virus Particle(A549) Virosomes (A549)
1.5 0.353 0.265
3.0 0.961 0.949
Neuraminidase activity of equivalent amounts of HN, comparing the native viral activity with cleaved and purified HN. The equivalent amounts of HN were incubated for 30 min at 37°C with 2.0 mg of N-acetylneuramin-lactose and then assayed for free sialic acid. HN represents -25% of total virion protein.
The data in this table shows that purified HN is recovered, with no loss of biological activity in the cleaved purified monomers.
Crystallization of Cleaved HN
The hanging drop method was used to crystallize the cleaved HN protein.
A mixture ofthe cleaved HN protein and precipitant was made, as described below, to be dropped on the crystallization surface. • One volume of purified cleaved HN protein (1 Omg/ml)
• One volume of H2O
• One volume of precipitant (20% Polyethylene glycol 4000 + 160mM (NH4)2SO4 + 80mM Acetate Buffer pH 4.6)
The drops were made over a well containing the precipitate. Crystals ranging in size from 0.2-0.7 mm were formed in 2-7 days (Fig. 2). Some of these crystals were removed, washed, and assayed for neuraminidase activity, which they were found to retain. Other washed crystals were run on a stained gel and found to migrate in the same position as the cleaved HN. As many as 40 crystals were observed in one drop and a number of different crystal forms were noted, including bipyramidal, rhomboid, and cubic. Initial X-ray analyses discussed in the next section indicates that the 0.2-0.25 mm crystal diffracts at moderately high resolution. Fewer crystals were also produced in a drop, but of much larger size, 0.4-0.6 mm.
Example 2: X-ray Diffraction Analysis
The first crystals produced (~0.2-0.25 mm) were X-ray analyzed on a rotating Cu anode X- ray source operating at 40 kV and 100 mA.
Figure 3 shows the diffraction pattem from a single frame of several hundred collected. Crystals were stable for at least 20 hrs. Frozen crystals were used for longer X-ray exposures (48 hrs), the crystals being stable to the X-rays in the frozen state. To collect the maximum number of useful reflections, multiple frames were collected as the crystal was rotated in the X-ray beam for 48 hrs. In this analysis, crystals diffracted to a resolution of 3.5 A (Fig. 4, edge). To increase the resolution further, slightly larger crystals (0.25 mm) were analyzed in a synchrotron high energy X- ray source. Using frozen crystals, X-ray diffraction data was collected every 6 minutes over a 24-hr period. A single frame is shown in Figure 5. The crystals diffracted to a relatively high resolution of 2.6 A. Example 3: Cloning and Sequencing of Nucleic Acid Encoding a Paramyxovirus HN Protein
HN gene of NDV was cloned by polymerase chain reaction (PCR) method using RNA extracted from vims infected BHK cells. First strand cDNA was synthesized by Moloney murine leukemia vims reverse transcriptase (Promega) using primer designed from consensus sequence found in NDV strains obtained from NIH GenBank. The synthesized cDNA was amplified by PCR using similarly designed primers. NDV HNcDNA containing full coding region was subcloned into plasmid pTFl (Takahashi et al, 1992, Bousse et al, 1994) at Hindlll and Kpnl sites.
Sequencing of the NDV HN cloned in pTFl was done by the dideoxy chain termination method using SEQUENASE version 2 DNA polymerase (US Biomedicals) following the manufacturers instructions. The primers used for sequence were designed from the sequence data of other NDV strains in NIH GenBank. The cDNA sequence is presented in Figure 4, and the corresponding amino acid sequence is presented in Figure 5. See, e.g., Takahashi et al, Genet. Anal. Tech. Appl. 9:91-95 (1992); Bousee et al, Virology 204:506-514. All references cited herein are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment ofthe present invention is disclosed, taught or suggested in the relevant art. The description ofthe specific embodiments will so fully reveal the general nature ofthe invention that others can, by applying knowledge within the skill ofthe art (including the contents ofthe references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept ofthe present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents ofthe disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology is to be interpreted by the skilled artisan in light ofthe teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: St. Jude Children's Research Hospital 332 North Lauderdale Memphis, TN 38105-2794 United States of America
(ii) TITLE OF INVENTION: Virus Protein Purification from
Virosomes
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox, P.L.L.C.
(B) STREET: 1100 New York Avenue, N , Suite 600
(C) CITY: Washington
(D) STATE: D.C.
(E) COUNTRY: USA
(F) ZIP: 20005-3934
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: To be assigned
(B) FILING DATE: Herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER: U.S. 60/003,447
(B) FILING DATE: 08-SEP-1995
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Fox, Samuel L.
(B) REGISTRATION NUMBER: 30,353
(C) REFERENCE/DOCKET NUMBER: 0656.054PC01
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 371-2600
(B) TELEFAX: (202) 371-2540
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1734 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1731
(xi) SEQUENCE DESCRIPTION: SEQ ID NO.l:
ATG GAC CGC GCA GTT AGC CAA GTT GCG TTA GAG AAT GAT GAA AGA GAG 48 Met Asp Arg Ala Val Ser Gin Val Ala Leu Glu Asn Asp Glu Arg Glu 1 5 10 15
GCA AAA AAT ACA TGG CGC TTG ATA TTC CGG ATT GCA ATC TTA CTC TTA 96 Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Leu Leu 20 25 30
ACA GTA GTG ACC TTA GCT ACA TCT GTA GCC TCC CTT GTA TAT AGC ATG 144 Thr Val Val Thr Leu Ala Thr Ser Val Ala Ser Leu Val Tyr Ser Met 35 40 45
GGG GCT AGC ACA CCT AGC GAC CTT GTA GGC ATA CCG ACC AGG ATT TCT 192 Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser 50 55 60
AGG GCA GAA GAA AAG ATT ACA TCT GCA CTT GGT TCC AAT CAA GAT GTA 240 Arg Ala Glu Glu Lys Ile Thr Ser Ala Leu Gly Ser Asn Gin Asp Val 65 70 75 80
GTA GAT AGG ATA TAT AAG CAA GTG GCC CTT GAG TCT CCG TTG GCA TTG 288 Val Asp Arg Ile Tyr Lys Gin Val Ala Leu Glu Ser Pro Leu Ala Leu 85 90 95
TTA AAC ACT GAG ACC ACA ATT ATG AAC GCA ATA ACA TCT CTC TCT TAT 336 Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser Leu Ser Tyr 100 105 110
CAG ATT AAT GGA GCT GCG AAC AAC AGC GGG TGG GGG GCA CCT ATC CAT 384 Gin Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro Ile His 115 120 125
GAC CCA GAT TTT ATC GGG GGG ATA GGC AAA GAA CTC GTT GTA GAT AAT 432 Asp Pro Asp Phe Ile Gly Gly Ile Gly Lys Glu Leu Val Val Asp Asn 130 135 140
GCT AGT GAT GTC ACA TCA TTC TAT CCC TCT GCA TTT CAA GAA CAT CTG 480 Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gin Glu His Leu 145 150 155 160
AAT TTT ATC CCG GCG CCT ACT ACA GGA TCA GGT TGC ACT CGG ATA CCT 528 Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro 165 170 175 TCA TTT GAC ATG AGT GCT ACC CAT TAC TGC TAC ACT CAT AAT GTA ATA 576 Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile 180 185 190
TTG TCT GGA TGC AGA GAT CAC TCA CAC TCA CAT CAG TAT TTA GCA CTT 624 Leu Ser Gly Cys Arg Asp His Ser His Ser His Gin Tyr Leu Ala Leu 195 200 205
GGT GTG CTC CGG ACA ACT GCA ACA GGG AGG ATA TTC TTT TCT ACT CTG 672 Gly Val Leu Arg Thr Thr Ala Thr Gly Arg Ile Phe Phe Ser Thr Leu 210 215 220
CGT TCC ATC AGT CTG GAT GAC ACC CAA AAT CGG AAG TCT TGC AGT GTG 720 Arg Ser Ile Ser Leu Asp Asp Thr Gin Asn Arg Lys Ser Cys Ser Val 225 230 235 240
AGT GCA ACT CCC TTA GGT TGT GAT ATG CTG TGC TCG AAA GTC ACG GAG 768 Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Val Thr Glu 245 250 255
ACA GAG GAA GAA GAT TAT AAC TCA GCT GTC CCT ACG CTG ATG GCA CAT 816 Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr Leu Met Ala His 260 265 270
GGG AGG TTA GGG TTC GAC GGC CAA TAC CAC GAA AAG GAC CTA GAC GTC 864 Gly Arg Leu Gly Phe Asp Gly Gin Tyr His Glu Lys Asp Leu Asp Val 275 280 285
ACA ACA TTA TTT GAG GAC TGG GTG GCC AAC TAC CCA GGA GTA GGG GGT 912 Thr Thr Leu Phe Glu Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly 290 295 300
GGA TCT TTT ATT GAC GGC CGC GTA TGG TTC TCA GTC TAC GGA GGG CTG 960 Gly Ser Phe Ile Asp Gly Arg Val Trp Phe Ser Val Tyr Gly Gly Leu 305 310 315 320
AAA CCC AAT TCA CCC AGT GAC ACT GTA CAG GAA GGG AAA TAC GTA ATA 1008 Lys Pro Asn Ser Pro Ser Asp Thr Val Gin Glu Gly Lys Tyr Val Ile 325 330 335
TAC AAG CGA TAC AAT GAC ACA TGC CCA GAT GAG CAA GAC TAC CAG ATC 1056 Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gin Asp Tyr Gin Ile 340 345 350
CGA ATG GCC AAG TCT TCG TAT AAG CCC GGG CGG TTT GGT GGG AAA CGC 1104 Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly Lys Arg 355 360 365
ATA CAG CAG GCT ATC TTA TCT ATC AAG GTG TCA ACA TCT TTG GGC GAA 1152 Ile Gin Gin Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu 370 375 380
GAC CCA GCA CTG ACT GTA CCG CCC AAC ACA GTC ACA CTC ATG GGG GCC 1200 Asp Pro Ala Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala 385 390 395 400 GAA GGA AGA ATT CTC ACA GTA GGG ACA TCT CAT TTC TTG TAT CAG CGA 1248 Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gin Arg 405 410 415
GGG TCA TCA TAC TTC TCT CCC GCG TTA TTA TAT CCT ATG ACA GTC AGC 1296 Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser 420 425 430
AAC AAA ACA GCC ACT CTT CAT AGT CCC TAT ACA TTC AAT GCC TTC ACT 1344 Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr 435 440 445
CGG CCA GGT AGT ATC CCT TGC CAG GCT TCA GCA AGA TGC CCC AAC TCG 1392 Arg Pro Gly Ser Ile Pro Cys Gin Ala Ser Ala Arg Cys Pro Asn Ser 450 455 460
TGT GTT ACT GGA GTC TAT ACA GAT CCA TAT CCC CTA ATC TTC TAT AGG 1440 Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe Tyr Arg 465 470 475 480
AAC CAC ACC TTG CGA GGG GTA TTC GGG ACA ATG CTT GAT AGT GAA CAA 1488 Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Ser Glu Gin 485 490 495
GCA AGA CTT AAT CCT GCG TCT GCA GTA TTC GAT AGC ACA TCC CGC AGT 1536 Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser 500 505 510
CGC ATA ACT CGA GTG AGT TCA AGC AGC ACC AAA GCA GCA TAC ACA ACA 1584 Arg Ile Thr Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr 515 520 525
TCA ACT TGT TTT AAA GTT GTC AAG ACC AAT AAG ACC TAT TGT CTC AGC 1632 Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser 530 535 540
ATT GCT GAA ATA TCT AAT ACT CTC TTC GGA GAA TTC AGA ATC GTC CCG 1680 Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro 545 550 555 560
TTA CTA GTT GAG ATC CTC AAA AAT GAT GGG GTT AGA GAA GCC AGG TCT 1728 Leu Leu Val Glu Ile Leu Lys Asn Asp Gly Val Arg Glu Ala Arg Ser 565 570 575
GGT TAG 1734
Gly
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 577 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Asp Arg Ala Val Ser Gin Val Ala Leu Glu Asn Asp Glu Arg Glu 1 5 10 15
Ala Lys Asn Thr Trp Arg Leu Ile Phe Arg Ile Ala Ile Leu Leu Leu 20 25 30
Thr Val Val Thr Leu Ala Thr Ser Val Ala Ser Leu Val Tyr Ser Met 35 40 45
Gly Ala Ser Thr Pro Ser Asp Leu Val Gly Ile Pro Thr Arg Ile Ser 50 55 60
Arg Ala Glu Glu Lys Ile Thr Ser Ala Leu Gly Ser Asn Gin Asp Val 65 70 75 80
Val Asp Arg Ile Tyr Lys Gin Val Ala Leu Glu Ser Pro Leu Ala Leu 85 90 95
Leu Asn Thr Glu Thr Thr Ile Met Asn Ala Ile Thr Ser Leu Ser Tyr 100 105 110
Gin Ile Asn Gly Ala Ala Asn Asn Ser Gly Trp Gly Ala Pro Ile His 115 120 125
Asp Pro Asp Phe Ile Gly Gly Ile Gly Lys Glu Leu Val Val Asp Asn 130 135 140
Ala Ser Asp Val Thr Ser Phe Tyr Pro Ser Ala Phe Gin Glu His Leu 145 150 155 160
Asn Phe Ile Pro Ala Pro Thr Thr Gly Ser Gly Cys Thr Arg Ile Pro 165 170 175
Ser Phe Asp Met Ser Ala Thr His Tyr Cys Tyr Thr His Asn Val Ile 180 185 190
Leu Ser Gly Cys Arg Asp His Ser His Ser His Gin Tyr Leu Ala Leu 195 200 205
Gly Val Leu Arg Thr Thr Ala Thr Gly Arg Ile Phe Phe Ser Thr Leu 210 215 220
Arg Ser Ile Ser Leu Asp Asp Thr Gin Asn Arg Lys Ser Cys Ser Val 225 230 235 240
Ser Ala Thr Pro Leu Gly Cys Asp Met Leu Cys Ser Lys Val Thr Glu 245 250 255
Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val Pro Thr Leu Met Ala His 260 265 270 Gly Arg Leu Gly Phe Asp Gly Gin Tyr His Glu Lys Asp Leu Asp Val 275 280 285
Thr Thr Leu Phe Glu Asp Trp Val Ala Asn Tyr Pro Gly Val Gly Gly 290 295 300
Gly Ser Phe Ile Asp Gly Arg Val Trp Phe Ser Val Tyr Gly Gly Leu 305 310 315 320
Lys Pro Asn Ser Pro Ser Asp Thr Val Gin Glu Gly Lys Tyr Val Ile 325 330 335
Tyr Lys Arg Tyr Asn Asp Thr Cys Pro Asp Glu Gin Asp Tyr Gin lie 340 345 350
Arg Met Ala Lys Ser Ser Tyr Lys Pro Gly Arg Phe Gly Gly Lys Arg 355 360 365
Ile Gin Gin Ala Ile Leu Ser Ile Lys Val Ser Thr Ser Leu Gly Glu 370 375 380
Asp Pro Ala Leu Thr Val Pro Pro Asn Thr Val Thr Leu Met Gly Ala 385 390 395 400
Glu Gly Arg Ile Leu Thr Val Gly Thr Ser His Phe Leu Tyr Gin Arg 405 410 415
Gly Ser Ser Tyr Phe Ser Pro Ala Leu Leu Tyr Pro Met Thr Val Ser 420 425 430
Asn Lys Thr Ala Thr Leu His Ser Pro Tyr Thr Phe Asn Ala Phe Thr 435 440 445
Arg Pro Gly Ser Ile Pro Cys Gin Ala Ser Ala Arg Cys Pro Asn Ser 450 455 460
Cys Val Thr Gly Val Tyr Thr Asp Pro Tyr Pro Leu Ile Phe Tyr Arg 465 470 475 480
Asn His Thr Leu Arg Gly Val Phe Gly Thr Met Leu Asp Ser Glu Gin 485 490 495
Ala Arg Leu Asn Pro Ala Ser Ala Val Phe Asp Ser Thr Ser Arg Ser 500 505 510
Arg Ile Thr Arg Val Ser Ser Ser Ser Thr Lys Ala Ala Tyr Thr Thr 515 520 525
Ser Thr Cys Phe Lys Val Val Lys Thr Asn Lys Thr Tyr Cys Leu Ser 530 535 540
Ile Ala Glu Ile Ser Asn Thr Leu Phe Gly Glu Phe Arg Ile Val Pro 545 550 555 560
Leu Leu Val Glu Ile Leu Lys Asn Asp Gly Val Arg Glu Ala Arg Ser 565 570 575
Gly

Claims

What Is Claimed Is:
1. A method for crystallizing a viral attachment protein (VAP) from a vims, comprising
(a) providing a purified virosome derived from said vims containing the VAP in membrane bound form;
(b) cleaving, with a proteolytic enzyme, the soluble portion of the VAP from the transmembrane portion of the VAP in said virosome, to provide a soluble form ofthe VAP as a cleaved VAP having biological activity; and
(c) crystallizing the cleaved VAP using a hanging drop vapor diffusion method, to provide crystallized cleaved VAP having biological activity.
2. A method according to claim 1 , wherein said proteolytic enzyme is selected from the group consisting of pronase, trypsin, thermolysin, protease K, pronase, papain, endoproteinase Lys- c, endoproteinase Gly-c, endoproteinase Asp-N, endoproteinase Arg-c, chymotrypsin, bromelain, carboxypeptidase y, carboxypeptidase P, carboxypeptidase A, carboxypeptidase B, aminopeptidase
M, pepsin, plasmin, and leucine aminopeptidase
3. A method according to claim 1 , wherein said VAP is hemagglutinin neuraminidase (HN).
4. A method according to claim 1, wherein said crystallization step is done under conditions of 5-100 mg/ml cleaved VAP; PEGl 00-8000; precipitating salt; buffered saline, and pH 4-9.
5. A method according to claim 1 , wherein said vims strain is of a paramyxovirus.
6. A method according to claim 5, wherein said paramyxovirus is selected from mumps, measles, parainfluenza vims (PIV), sendai vims (SV) and Newcastle disease vims (NDV).
7. A method according to claim 6, wherein the strain of the paramyxovirus is the Kansas strain of Newcastle disease vims (NDV).
8. A method according to claim 7, wherein the crystallization conditions are one volume of 5-00 mg/ml of cleaved HN; one volume of water; and one volume of 20-25% PEG3350-4000,
160-200 mM ammonium sulfate and 80-100 mM acetate buffered saline (pH 4.2-4.8).
9. A method according to claim 1 , wherein the cleaved VAP crystals have biological activity and provide a wider x-ray crystallograph of about 5-3.5 A.
10. A method according to claim 9, wherein the resolution is 2.6 A.
11. A crystallized cleaved VAP , provided by a method according to claim 1.
12. A crystallized cleaved VAP according to claim 11 , wherein the crystals are of an HN of the Kansas strain of NDV.
13. A paramyxovirus HN protein, comprising the amino acid sequence:
APIHDP DFIGGIGKEL WDNASDVTS
FYPSAFQEHL NFIPAPTTGS GCTRIPSFDM SATHYCYTHN VILSGCRDHS HSHQYLALGV LRTTATGRIF FSTLRSISLD DTQNRKSCSV SATPLGCDML CSKVTETEEE DYNSAVPTLM AHGRLGFDGQ YHEKDLDVTT LFEDWVANYP GVGGGSFIDG RVWFSVYGGL KPNSPSDTVQ EGKYVIYKRY NDTCPDEQDY QIRMAKSSYK PGRFGGKRIQ QAILSIKVST SLGEDPALTV PPNTVTLMGA
EGRILTVGTS HFLYQRGSSY FSPALLYPMT VSNKTATLHS PYTFNAFTRP GSIPCQASAR CPNSCVTGVY TDPYPLIFYR NHTLRGVFGT MLDSEQARLN PASAVFDSTS RSRITRVSSS STKAAYTTST CFKWKTNKT YCLSIAEISN TLFGEFRIVP LLVEILKNDG VREARSG
14. A paramyxovirus HN protein according to claim 13, comprising the amino acid sequence: 1 MDRAVSQVAL ENDEREAKNT WRLIFRIAIL LLTWTLATS VASLVYSMGA
51 STPSDLVGIP TRISRAEEKI TSALGSNQDV VDRIYKQVAL ESPLALLNTE
101 TTIMNAITSL SYQINGAANN SGWGAPIHDP DFIGGIGKEL WDNASDVTS
151 FYPSAFQEHL NFIPAPTTGS GCTRIPSFDM SATHYCYTHN VILSGCRDHS 201 HSHQYLALGV LRTTATGRIF FSTLRSISLD DTQNRKSCSV SATPLGCDML
251 CSKVTETEEE DYNSAVPTLM AHGRLGFDGQ YHEKDLDVTT LFEDWVANYP
301 GVGGGSFIDG RVWFSVYGGL KPNSPSDTVQ EGKYVIYKRY NDTCPDEQDY
351 QIRMAKSSYK PGRFGGKRIQ QAILSIKVST SLGEDPALTV PPNTVTLMGA
401 EGRILTVGTS HFLYQRGSSY FSPALLYPMT VSNKTATLHS PYTFNAFTRP 451 GSIPCQASAR CPNSCVTGVY TDPYPLIFYR NHTLRGVFGT MLDSEQARLN
501 PASAVFDSTS RSRITRVSSS STKAAYTTST CFKWKTNKT YCLSIAEISN
551 TLFGEFRIVP LLVEILKNDG VREARSG
15. An antibody which specifically binds an epitope of at least 4 amino acids of a VAP according to claim 11.
16. A host cell, comprising nucleic acid which encodes an antibody according to claim 15.
17. A nucleic acid molecule, corresponding or complementary to at least 15 nucleotides ofa DNA sequence encoding at least 5 amino acids ofthe amino acid sequence of a VAP of claim 11.
18. A nucleic acid molecule according to claim 17, comprising at least 15 nucleotides of Figure 5.
19. A nucleic acid according to claim 18, having the nucleotide sequence of Figure 5.
20. A vector, comprising a nucleic acid molecule according to claim 17.
21. A host, comprising a nucleic acid molecule according to claim 17.
PCT/US1996/014187 1995-09-08 1996-09-06 Virus protein purification from virosomes WO1997009345A1 (en)

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EP2193808A1 (en) 1999-08-21 2010-06-09 Nycomed GmbH Synergistic combination
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2193808A1 (en) 1999-08-21 2010-06-09 Nycomed GmbH Synergistic combination
WO2002010459A2 (en) * 2000-07-27 2002-02-07 Biocryst Pharmaceuticals, Inc. Three dimensional structure of paramyxovirus hemagglutinin-neuraminidases and use thereof
WO2002010459A3 (en) * 2000-07-27 2003-02-27 Biocryst Pharm Inc Three dimensional structure of paramyxovirus hemagglutinin-neuraminidases and use thereof
US7772318B2 (en) 2005-04-04 2010-08-10 Rohm And Haas Company Aqueous polymer dispersions

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