WO2022150293A1 - Coronavirus replicon delivery particles and methods of use thereof - Google Patents

Coronavirus replicon delivery particles and methods of use thereof Download PDF

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Publication number
WO2022150293A1
WO2022150293A1 PCT/US2022/011115 US2022011115W WO2022150293A1 WO 2022150293 A1 WO2022150293 A1 WO 2022150293A1 US 2022011115 W US2022011115 W US 2022011115W WO 2022150293 A1 WO2022150293 A1 WO 2022150293A1
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cell
nucleic acid
replicon
cells
coronavirus
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PCT/US2022/011115
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French (fr)
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Charles Rice
Inna RICARDO-LAX
Joseph M. Luna
Nadine EBERT
Jörg JORES
Fabien LABROUSSAA
Volker Thiel
Thi Nhu Thao TRAN
John T. POIRIER
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The Rockefeller University
New York University
The University Of Bern
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Publication of WO2022150293A1 publication Critical patent/WO2022150293A1/en

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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20023—Virus like particles [VLP]
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20031—Uses of virus other than therapeutic or vaccine, e.g. disinfectant
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to coronavirus reporter replicons and method of use thereof and more specifically to trans-packaged coronavirus reporter replicons and method of use thereof.
  • SARS-CoV-2 the causative agent of COVID-19, is a member of the Coronaviridae family of positive sense ssRNA viruses. Similar to SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV), the pathogenicity of SARS-CoV-2 necessitates that molecular virology studies of infectious virus can only occur in high containment biological safety level 3 (BSL3) laboratory settings. While studies of viral components in BSL2 settings have been invaluable to establish initial lists of putative host factors and to refine viral enzymatic mechanisms, non-infectious systems permitting the study of intracellular replication have been lacking.
  • BSL3 containment biological safety level 3
  • RNAs Self-replicating RNAs, known as replicons, have provided an important model system to study numerous aspects of RNA virus life cycles.
  • Replicons are typically constructed using reverse genetics systems to replace one or more viral structural proteins with selectable or reporter genes.
  • the replicon RNA Upon transfection or electroporation of viral replicon RNA into cells, the replicon RNA is translated to produce viral enzymes and cofactors necessary to establish RNA replication factories, with reporter genes providing convenient readouts for replicon viability and for the selection of non-cytopathic variants.
  • reporter genes providing convenient readouts for replicon viability and for the selection of non-cytopathic variants.
  • RNA replication, translation, and functions of viral gene products proceed without producing infectious virus.
  • Such systems have been invaluable as molecular virology and high-throughput compound screening and drug development platforms, most notably with hepatitis C virus.
  • RNA replicons e.g., SARS-CoV-2 RNA replicons
  • trans-packaged replicons TPRs
  • RDPs replicon delivery particles
  • this disclosure provides a nucleic acid molecule comprising or encoding a coronavirus replicon.
  • the replicon comprises: (i) a genomic or subgenomic nucleotide sequence of a coronavirus, wherein the nucleotide sequence comprises at least one of the coding sequences of a membrane (M) protein of the coronavirus and an envelope (E) protein of the coronavirus and wherein the coding sequence of a spike (S) protein of the coronavirus is inactivated or deleted; and (ii) a second nucleotide sequence encoding a selectable marker suitable for selection, wherein the selectable marker is under the control of the RNA virus replication machinery.
  • the replicon lacks the Spike-coding sequence, which can be replaced by a reporter gene cassette to monitor replicon activity.
  • examples include Trans- packaged Spike deleted Nspl mutant replicon (TPR AS+) in which Nspl is modified to inhibit its activity, Trans-packaged Spike deleted Minus Accessory replicon (TPR AAcc) which lacks all accessory genes, Trans-packaged Spike deleted Minus SEM replicon (TPR ASEM) which lacks all structural genes, and Trans-packaged Spike deleted minimal replicon (miniTPR) which lacks all accessory and structural genes.
  • the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • the coding sequence of the spike protein or a portion thereof is replaced with the second nucleotide sequence.
  • the selectable marker is a gene that confers resistance to an antibiotic.
  • the nucleic acid molecule further comprises a reporter gene.
  • the reporter gene is selected from the group consisting of NeonGreen, gaussia luciferase (Glue), mScarlet, green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced
  • GFP GFP
  • EGFP enhanced CFP
  • EYFP enhanced YFP
  • GFPS65T Emerald, Venus, mOrange, Topaz
  • GFPuv destabilized EGFP
  • dEGFP destabilized ECFP
  • dEYFP destabilized EYFP
  • HcRed HcRed
  • t-HcRed DsRed
  • DsRed2 t-dimer2(12)
  • mRFPl pocilloporin
  • Renilla GFP Monster GFP
  • paGFP Kaede protein
  • Kaede protein a Phycobiliprotein
  • a biologically active variant or fragment of thereof a biologically active variant or fragment of thereof.
  • the reporter gene is operatively linked to a spike transcription regulating sequence (TRS). In some embodiments, the reporter gene is operatively linked to the TRS through a T2A self-cleaving sequence.
  • TRS spike transcription regulating sequence
  • the nucleic acid molecule has at least 80% sequence identity to SEQ ID NO: 1 or 3, or comprises the nucleic acid sequence of SEQ ID NO: 1 or 3.
  • this disclosure also provides a virus particle or a virus-like particle comprising a nucleic acid molecule described above.
  • the virus particle or virus-like particle comprises a vesicular stomatitis virus G (VSV-G) protein or a variant/fragment thereof.
  • this disclosure additionally provides a cell or cell line comprising a nucleic acid molecule described above.
  • the cell or cell line further comprises a second nucleic acid molecule comprising a coding sequence of a VSV-G protein or a variant/fragment thereof.
  • the VSV-G protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or comprises the amino acid sequence of SEQ ID NO: 2.
  • VSV-G protein sequence SEQ ID NO: 2
  • the cell or cell line further comprises a third nucleic acid molecule comprising a coding sequence of a Spike protein or a variant/fragment thereof
  • the Spike protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or comprises the amino acid sequence of SEQ ID NO: 4.
  • the cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the cell is a Huh-7.5 cell or a BHK-21 cell. In some embodiments, the cell is a lung organoid.
  • composition comprising a nucleic acid molecule or a cell or cell line, as described above, and a pharmaceutically acceptable carrier.
  • this disclosure provides a kit comprising a nucleic acid molecule, a cell or cell line, or a composition, as described above.
  • this disclosure further provides a method of preparing a coronavirus replicon-harbored cell.
  • the method comprises: (i) introducing a nucleic acid molecule described above to a cell; and (ii) culturing the cell in a cell culture medium to produce the coronavirus replicon-harbored cell.
  • the cell can contain a second nucleic acid comprising a coding sequence of the VSV-G protein or a variant/fragment thereof.
  • the method may further comprise introducing to the cell a second nucleic acid comprising a coding sequence of the VSV-G protein or a variant/fragment thereof.
  • the method may further comprise introducing to the cell a third nucleic acid comprising a coding sequence of the Spike protein or a variant/fragment thereof.
  • the cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the cell is a Huh-7.5 cell.
  • this disclosure further provides a method for screening for antiviral agents for a coronavirus.
  • the method comprises: (i) contacting a cell or cell line described above with a candidate agent; and (ii) determining an increase or decrease in replication or activity of the coronavirus virus replicon relative to a control cell or cell line harboring the same replicon, wherein the control cell or cell line has not been contacted with the candidate agent.
  • the coronavirus is SARS-CoV or SARS-CoV-2.
  • the step of determining comprises determining a level of production of a coronavirus protein or a coronavirus RNA transcript.
  • the candidate agent comprises an organic compound or an antisense nucleic acid.
  • FIGS. 1A, IB, 1C, ID, IE, IF, and 1G are a set of diagrams showing SARS-CoV-2 replicon design and launch optimization.
  • FIG. 1A is a schematic of a modular SARS-CoV-2 replicon design. Fragments from (18) are shown in orange, and fragments harboring mutations in nspl or the RdRP (nspl2) are shown in red. Fragments to encode a spike-replaced reporter gene cassette, accessory proteins, and flanking regions are shown in purple, green, or blue, respectively.
  • FIG. IB shows agarose gel of replicon DNA recovered from yeast or bacteria, before or after amplification with phi29 DNA polymerase.
  • FIG. 1C shows agarose gel of T7 RNA transcription reactions from DNA plasmids shown in FIG. IB. Arrow highlights the expected size of full-length RNA. Asterisk denotes a non-specific band.
  • FIG. IF shows a flowchart diagram of optimized RNA production for SARS-CoV-2 replicons. Viral fragments and reporter transgenes are cloned and assembled in yeast. Yeast derived plasmids can either be propagated in bacteria or in yeast, in which case they are treated with plasmid-safe DNAse to remove DNA contaminants. Subsequent phi29 MDA amplification ensures full-length DNA template availability for T7 transcription.
  • FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21, and 2J are a set of diagrams showing that SARS- CoV-2 replicons are sensitive to antiviral compounds, required host factor loss, and viral mutant phenotypes.
  • FIG. 2A shows cumulative timecourse measurements of gaussia luciferase (Glue) in the supernatants of Huh-7.5 cells electroporated with the Glue replicon and seeded with 100 nM remdesivir (Rem) or vehicle.
  • a polymerase deficient (pol-) replicon was used as negative control. Dashed line indicates the limit of detection. Error bars represent standard deviation of 3 biological repeats.
  • FIG. 1A shows cumulative timecourse measurements of gaussia luciferase (Glue) in the supernatants of Huh-7.5 cells electroporated with the Glue replicon and seeded with 100 nM remdesivir (
  • FIG. 2B shows qRT-PCR measurements for subgenomic N RNA in Huh-7.5 cells harboring Glue replicons electroporated with the Glue replicon and seeded with lOOnM remdesivir (Rem) or vehicle.
  • a polymerase deficient (pol-) replicon was used for normalization (dashed line). Error bars represent standard deviation of 3 biological repeats.
  • FIG. 2C shows that Huh-7.5 Cells were electroporated with the Glue replicon and seeded in 96-wells plates on a dose-curve of remdesivir.
  • GLuc signal was measured in sup (black circles), and cell viability was measured by cell-titer Glo assay (empty circles). Data was normalized to vehicle (DMSO)-treated cells. Error bars represent standard deviation of 5 biological repeats.
  • FIG. 2D is the same as FIG. 2C except that the cells were treated with Masitinib. Error bars represent standard deviation of 4 biological repeats.
  • FIG. 2E is the same as FIG. 2C except that the cells were treated with 27-hydroxycholesterol (27HC). Error bars represent standard deviation of 3 biological repeats.
  • FIG. 2F shows that WT or TMEM41B KO cells were electroporated with the GLuc replicon and seeded with lOOnM Remdesivir or vehicle.
  • GLuc was measured 24h post electroporation. Error bars represent standard deviation of 4 biological repeats.
  • FIG. 2G shows that cells in F were electroporated with SinRep-GFP Sindbis virus replicon RNA and seeded in 6-wells plates. Percent of GFP-positive cells was determined by flow cytometry 24h post electroporation. Error bars represent standard deviation from three biological replicates.
  • FIG. 2H is the same as FIG.
  • FIG. 21 shows that Huh7.5 cells were electroporated with either WT or NSP1 K164A/H165A mutant (NSPl ' ). Electroporated cells were seeded with lOOnM Remdesivir or vehicle, and GLuc levels on the supernatant were measured at the indicated time points. Error bars represent standard deviation of 4 biological repeats.
  • FIG. 2J is the same as FIG. 21 except that cell viability was measured at each time point by cell-titer Glo assay. Mock-electroporated cells were used as control for normal post-electroporation cell viability.
  • FIGS. 3A, 3B, 3C, 3D, 3E, and 3F are a set of diagrams showing kinetics and drug susceptibility of different replicons.
  • FIG. 3 A shows that Huh7.5 cells were electroporated with WT or NSP- replicons and seeded on a dose-curve of Interferon alpha (IFNa). GLuc activity (closed circles) and cell viability (open circles) were measured 24 hours post electroporation.
  • FIG. 3B is the same as FIG. 3A except that cells were treated with interferon beta (IFNp)
  • FIG. 3C is the same as FIG. 3A except that cells were treated with Remdesivir.
  • FIG. 3 A shows that Huh7.5 cells were electroporated with WT or NSP- replicons and seeded on a dose-curve of Interferon alpha (IFNa). GLuc activity (closed circles) and cell viability (open circles) were measured 24 hours post electroporation.
  • FIG. 3D is a schematic of the full replicon, containing most structural proteins excluding Spike and all the accessory proteins, and minireplicons, which are devoid of all accessory and structural proteins and encode only for the replicase and N genes.
  • FIG. 3E shows kinetics of GLuc expression of the full and mini replicons. Huh7.5 cells were electroporated with the full or mini replicons and seeded with 100 nM Remdesivir (open circles) or vehicle (closed circles). GLuc activity in the supernatant was measured at the indicated time points.
  • FIG. 3F is the same as FIG. 3E, except that cell viability was measured by cell titer Glo assay and normalized to day 1 post electroporation.
  • FIGS. 4A, 4B, 4C, and 4D are a set of diagrams showing replicon delivery by transpackaging with VSV-G.
  • FIG. 4 A shows an experimental scheme describing the trans-packaging of replicons with VSV-G. Briefly, BHK-21 cells were transfected with VGV-G, and the next day electroporated with the full SARS-CoV2 replicon. After 48h, the supernatant of the producer cells was collected and concentrated. Multiple cell types can be transduced with the resulting trans- packaged replicons (TRPs).
  • TRPs trans- packaged replicons
  • FIG. 4B shows that indicated cells were seeded in 96-well plates and transduced with TPR-NeonGreen, produced as described in FIG. 4A. Brightfield and fluorescent images were taken at xlO magnification 24h after transduction.
  • FIG. 4C shows that Huh7.5 cells were transduced with concentrated or 1:10 diluted TPR-NeonGreen, and percent of reporter positive cells was measured by flow cytometry. As negative controls, cells were transduced with similarly collected supernatants from cells that were electroporated with the minireplicon instead, or where VSV-G was replaced by a control plasmid.
  • FIG. 5E shows that TPRs are single-cycle infectious virions. Huh7.5 cells were transduced with IOOmI of TPRs per 2ml in a 6-well format (P0 supernatant). After lhr, inoculum was saved and concentrated with PEG (P0’ supernatant).
  • FIGS. 5A, 5B, and 5C are a set of photographs and diagrams showing replicon assembly and RNA validation (Related to FIG. 1).
  • FIG. 5A shows that six different replicons were assembled in yeast, and DNA from four colonies of each assembly were extracted and subjected to multiplex PCR with primer set #1 (see Table 1). Expected PCR product sizes are indicated on the right.
  • FIG. 5B shows that RNA was in-vitro transcribed from lug of the indicated phi-29 amplified DNA templates, using T7 RiboMAX Express Large Scale RNA Production System (Promega) or HiScribe T7 High Yield RNA Synthesis kit (NEB).
  • FIG. 5C shows the results of qRT-PCR measurements for N RNA in Huh-7.5 cells electroporated with the 5pg Glue replicon RNA plus 2pg N mRNA and seeded with lOOnM remdesivir (Rem) or vehicle. The signal from mock infected cells was used for normalization (dashed line). Error bars represent standard deviation of 3 biological repeats.
  • FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H are a set of diagrams showing susceptibility of the Spike-deleted and minireplicons to antiviral compounds (Related to FIG. 3).
  • FIGS. 6A, 6B, 6C, and 6D show that Huh7.5 cells were electroporated with GLuc spike-deleted replicon and seeded in 96-wells plate on a dose-curve of Remdesivir (FIG. 6A), IFNa (FIG. 6B) IFNb (FIG. 6C) or AM580 (FIG. 6D).
  • GLuc activity in the supernatant (closed circles) and cell viability (open circles) were measured 48h post electroporation. Error bars represent standard error from 3 biological repeats.
  • FIGS. 7A, 7B, 7C, 7D, 7E, and 7F are a set of diagrams showing trans-complementation of replicons with Spike yields single-cycle SARS-CoV-2.
  • FIG. 7A shows a scheme to trans complement replicons with ectopically expressed Spike for single-cycle virion production.
  • BHK- 21 cells are transfected with a Spike encoding plasmid, and 24 hours later electroporated with the full SARS-CoV2 replicon.
  • Supernatant from these producer cells (P0) is collected and passaged onto recipient cells (PI) yielding reporter activity.
  • a second round of passaging onto naive recipient cells (P2) fails to propagate the replicon.
  • FIG. 7A shows a scheme to trans complement replicons with ectopically expressed Spike for single-cycle virion production.
  • BHK- 21 cells are transfected with a Spike encoding plasmid, and 24 hours later
  • FIG. 7B shows a spike trans-packaged replicon consisting of the full Spike deleted replicon RNA alongside plasmid driven Spike expression. Nspl mutations relative to the WT sequence indicated.
  • FIG. 7C shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer cells (P0) with supernatants concentrated and subsequently passaged twice onto Huh-7.5 ACE2/TMPRSS2 (Huh-7.5 AT) cells (PI and P2). Immunofluorescence images at 4X of the mNeongreen signal (green) and N antibody staining (magenta) are shown. Scale bar, lOOpm.
  • FIG. 7D shows neongreen quantification per passage for the results in (FIG. 1C).
  • FIGS. 8A, 8B, 8C, 8D, and 8E are a set of diagrams showing ectopic Spike expression and quantification.
  • FIG. 8A shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer BHK-21 cells (P0) with supernatants subsequently passaged onto Huh-7.5 ACE2/TMPRSS2 (Huh-7.5 AT) cells (PI). Immunofluorescence images at 4X of the NeonGreen signal (green) and Spike C144 antibody staining (magenta) are shown. Scale bar, 100 pm.
  • FIG. 8A shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer BHK-21 cells (P0) with supernatants subsequently passaged onto Huh-7.5 ACE2/TMPRSS2 (Hu
  • FIG. 8C shows N immunofluorescence quantification per passage for the results in Figure 4C.
  • FIG. 8D shows immunofluorescence images N protein (magenta) in Huh-7.5 cells infected with SARS-CoV-2 (WA1/2020, left) or transduced with TPRs packaged with WT Spike (right). Images at 4X magnification, scale bar 500pm.
  • FIG. 8E shows quantification of results in (FIG. 8D), measuring approximate per cell area for the N protein signal between virus or TPR positive cells.
  • FIGS. 9A, 9B, and 9C are a set of diagrams showing neutralization assays with TPRs recapitulate authentic SARS-CoV-2 antibody phenotypes.
  • FIGS. 9A, 9B, and 9C show neutralization assays for SARS-CoV-2 TPRs or virus in the presence of increasing concentrations of antibodies.
  • WT Spike with Cl 44 antibody (FIG. 9A)
  • B.1.351 Spike with Cl 44 antibody (FIG. 9B)
  • B.1.351 with C135 antibody FIG. 9C) are shown.
  • N 3
  • error bars SEM.
  • RNA replicons e.g, SARS-CoV-2 RNA replicons
  • SARS-CoV-2 RNA replicons RNA replicons
  • the disclosed RNA replicons are broadly amenable to molecular virology studies and drug development screening efforts.
  • this disclosure provides a nucleic acid molecule comprising or encoding a coronavirus replicon.
  • the replicon comprises: (i) a genomic nucleotide sequence of a coronavirus, wherein the nucleotide sequence comprises at least one of the coding sequences of a membrane (M) protein of the coronavirus and an envelope (E) protein of the coronavirus and wherein the coding sequence of a spike (S) protein of the coronavirus is inactivated or deleted; and (ii) a second nucleotide sequence encoding a selectable marker suitable for selection, wherein the selectable marker is under the control of the RNA virus replication machinery.
  • the selectable marker is a gene that confers resistance to an antibiotic.
  • the coding sequence of the spike protein or a portion thereof is replaced with the second nucleotide sequence.
  • the nucleic acid molecule further comprises a reporter gene.
  • the reporter gene can be selected from the group consisting of NeonGreen, gaussia luciferase (Glue), green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Venus, mOrange, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t- HcRed, DsRed, DsRed2, t-dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein a Phycobiliprotein, and a biologically active variant or fragment
  • the reporter gene is operatively linked to a spike transcriptionregulating sequence (TRS).
  • TRS spike transcriptionregulating sequence
  • the reporter gene can be directly or indirectly linked to a linker or a cleavage site.
  • the reporter gene can be operatively linked to a TRS through a T2A self-cleaving sequence.
  • the nucleic acid molecule has at least 80% sequence identity to SEQ ID NO: 1 or 3 (listed below), or comprises the nucleic acid sequence of SEQ ID NO: 1 or 3.
  • gagtacactg actttgcaac atcagcttgt gttttggctg ctgaatgtac aatttttaaa
  • 11221 atattgggta gtgctttatt agaagatgaa tttacacctt ttgatgttgt tagacaatgc 11281 tcaggtgtta ctttccaaag tgcagtgaaa agaacaatca agggtacaca ccactggttg
  • gagtttttcc cacggtggat atttcttctt gcgctgagcg taagagctat ctgacagaac
  • RNA replicon or “replicon RNA” refer to RNA, which contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell.
  • the RNA molecule (1) encodes polymerase, replicase, or other proteins which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and (2) contain cis-acting
  • RNA sequences required for replication and transcription of the subgenomic replicon-encoded RNA may be bound during the process of replication to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components.
  • a coronavims-derived replicon RNA molecule typically contains the following ordered elements: 5’ viral or defective-interfering RNA sequence(s) required in cis for replication, sequences coding for biologically active coronavirus nonstructural proteins (e.g., nsPl, nsP2, nsP3, and nsP4), promoter for the subgenomic RNA, 3’ viral sequences required in cis for replication, and a polyadenylate tract.
  • RNA replicon generally refers to a molecule of positive polarity or “message” sense, and the RNA replicon may be of length different from that of any known, naturally-occurring coronavirus.
  • the RNA replicon does not contain the sequences of at least a spike protein.
  • the coding sequence of the spike protein can be substituted with heterologous sequences.
  • the RNA replicon may be packaged into a recombinant coronavirus particle, and it may include one or more sequences, such as packaging signals, which serve to initiate interactions with coronavirus structural proteins that lead to particle formation.
  • “subgenomic RNA” refers to an RNA molecule of a length or size which is smaller than the genomic RNA from which it was derived.
  • the coronavirus subgenomic RNA should be transcribed from an internal promoter, whose sequences reside within the genomic RNA or its complement. Transcription of a coronavirus subgenomic RNA may be mediated by the viral- encoded polymerase(s) associated with host cell-encoded proteins, ribonucleoprotein(s), or a combination thereof.
  • the subgenomic RNA is produced from a modified RNA replicon, as disclosed herein.
  • a part or the entire coding sequence for the spike protein is absent and/or modified in the nucleic acid molecules disclosed herein.
  • the modified coronavirus genome or RNA replicon can be devoid of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the sequence encoding the spike protein.
  • the modified coronavirus genome or RNA replicon is devoid of a substantial portion of or the entire sequence encoding the spike protein.
  • a “substantial portion” of a nucleic acid sequence comprises enough of the nucleic acid sequence encoding the viral structural protein to afford putative identification of that protein, either by manual evaluation of the sequence by one skilled in the art or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, Altschul SF el al. 1993, supra).
  • the modified coronavirus genome or RNA replicon is devoid of the entire sequence encoding the spike protein.
  • the nucleic acid molecules may include a modified coronavirus genome or RNA replicon containing one or more attenuating mutations so as to increase the safety of virus manipulation and/or administration.
  • attenuating mutation means a nucleotide mutation or an amino acid encoded in view of such mutation, which results in a decreased probability of causing disease in its host (i.e., a loss of virulence), in accordance with standard terminology in the art, whether the mutation is a substitution mutation or an in-frame deletion or insertion mutation. Attenuating mutations may be in the coding or non-coding regions of the coronavirus genome.
  • Attenuating mutation excludes mutations or combinations of mutations that would be lethal to the virus. Further, those skilled in the art will appreciate that some attenuating mutations may be lethal in the absence of a second-site suppressor mutation(s).
  • the nucleic acid molecules are recombinant nucleic acid molecules.
  • the term “recombinant” means any molecule (e.g., DNA, RNA, polypeptide) that results from human manipulation.
  • a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector.
  • a recombinant nucleic acid molecule (1) can be synthesized or modified in vitro , for example, using chemical or enzymatic techniques (e.g., by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site- specific recombination) of nucleic acid molecules; (2) may include conjoined nucleotide sequences that are not conjoined in nature; (3) can be engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and/or (4) may be manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.
  • chemical or enzymatic techniques
  • the nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis.
  • the nucleic acid molecules may include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and/or substituted in such a manner that such modifications provide the desired property in effecting a biological activity as described herein.
  • a nucleic acid molecule including a variant of a naturally-occurring nucleic acid sequence, can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et ah, In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).
  • sequence of a nucleic acid molecule can be modified with respect to a naturally-occurring sequence from which it is derived using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as but not limited to site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including chemical synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules, and combinations thereof.
  • classic mutagenesis techniques and recombinant DNA techniques such as but not limited to site-directed mutagenesis
  • chemical treatment of a nucleic acid molecule to induce mutations
  • Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or the replicon encoded by the nucleic acid molecule and/or by hybridization with a wild-type gene or fragment thereof, or by PCR using primers having homology to a target or wild-type nucleic acid molecule or sequence.
  • the modified coronavirus genome or RNA replicon is operably linked to a heterologous regulatory element.
  • regulatory element refers to a nucleotide sequence located upstream (5’), within, or downstream (3’) of a coding sequence such as, for example, a polypeptide-encoding sequence or a functional RNA-encoding sequence. Transcription of the coding sequence and/or translation of an RNA molecule resulting from transcription of the coding sequence is typically affected by the presence or absence of the regulatory element.
  • These regulatory elements may comprise promoters, cis-elements, enhancers, terminators, or introns.
  • the heterologous regulatory element is, or comprises, a promoter sequence.
  • the heterologous promoter sequence can be any heterologous promoter sequence, for example, a SP6 promoter, a T3 promoter, or a T7 promoter, or a combination thereof.
  • the promoter sequence includes a T7 promoter sequence.
  • the modified coronavirus genome or RNA replicon can include one or more heterologous transcriptional termination signal sequences.
  • transcriptional termination signal refers to a regulatory section of genetic sequence that causes RNA polymerase to cease transcription.
  • the heterologous transcriptional termination signal sequences can generally be any heterologous transcriptional termination signal sequences, and for example, a SP6 termination signal sequence, a T3 termination signal sequence, a T7 termination signal sequence, or a variant thereof.
  • the nucleic acid molecules can include at least one of the one or more heterologous transcriptional termination signal sequences selected from the group consisting of a SP6 termination signal sequence, a T3 termination signal sequence, a T7 termination signal sequence, or a variant thereof.
  • the transcriptional termination sequence includes a T7 termination signal sequence.
  • the nucleic acid molecules disclosed herein can include one or more expression cassettes.
  • the nucleic acid molecules can include at least two, at least three, at least four, at least five, or at least six expression cassettes.
  • expression cassette refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. The expression cassette may be inserted into a vector for targeting a desired host cell and/or into a subject.
  • expression cassette may be used interchangeably with the term “expression construct.”
  • expression cassette refers to a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and/or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene.
  • operably linked denotes a functional linkage between two or more sequences. For example, an operably linkage between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is a functional link that allows for expression of the polynucleotide of interest.
  • operably linked refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest.
  • operably linked denotes a configuration in which a regulatory sequence is placed at an appropriate position relative to a sequence that encodes a polypeptide or functional RNA such that the control sequence directs or regulates the expression or cellular localization of the mRNA encoding the polypeptide, the polypeptide, and/or the functional RNA.
  • a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence.
  • Operably linked elements may be contiguous or non-contiguous.
  • the techniques for operably linking two or more sequences of DNA together are familiar to the skilled worker, and such methods have been described in a number of texts for standard molecular biological manipulation (see, for example, Maniatis et ah, “Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Gibson et al, Nature Methods 6:343-45, 2009).
  • the disclosed nucleic acid molecules may include a codon- optimized sequence.
  • the nucleic acid sequence may be codon-optimized for expression in a eukaryote or eukaryotic cell.
  • the codon-optimized nucleic acid sequence is codon-optimized for operability in a eukaryotic cell or organism, e.g. , a yeast cell, or a mammalian cell or organism, including a mouse cell, a rat cell, and a human cell or non-human eukaryote organism.
  • codon optimization refers to a process of modifying a nucleic acid sequence to enhance expression in the host cells by substituting at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • Codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., el al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa ).
  • one or more codons in a sequence encoding a DNA/RNA-targeting IL-2 variant corresponds to the most frequently used codon for a particular amino acid.
  • codon usage in yeast reference is made to the online Yeast Genome database available at http://www.yeastgenome.org/community/codonusage.shtml, or Codon selection in yeast , Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31.
  • codon usage in plants including algae reference is made to Codon usage in higher plants, green algae, and cyanobacteria , Campbell and Gowri, Plant Physiol. 1990 January; 92(1): 1-11.; as well as Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan. 25; 17(2):477-98; or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton B R, J Mol Evol. 1998 April; 46(4):449-59.
  • Coronavirus refers to a genus in the family Coronaviridae, which family is in turn classified within the order Nidovirales.
  • the coronaviruses are large, enveloped, positive- stranded RNA viruses. They have the largest genomes of all RNA viruses and replicate by a unique mechanism that results in a high frequency of recombination.
  • the coronaviruses include antigenic groups I, II, and III.
  • coronaviruses include SARS coronavirus (i.e ., SARS-CoV, SARS-CoV-2), MERS coronavirus, transmissible gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, murine hepatitis virus, sialodacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as chimeras of any of the foregoing.
  • SARS coronavirus i.e ., SARS-CoV, SARS-CoV-2
  • MERS coronavirus transmissible gastroenteritis virus (TGEV)
  • human respiratory coronavirus porcine respiratory coronavirus
  • canine coronavirus canine coron
  • the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • this disclosure also provides a virus particle or a virus-like particle comprising a nucleic acid molecule described above.
  • the virus particle or virus-like particle comprises a vesicular stomatitis virus G (VSV-G) protein.
  • VSV-G vesicular stomatitis virus G
  • VLP refers to a nonreplicating, viral shell.
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art and discussed more fully below.
  • VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical and immunological characterizations, and the like. See , e.g., Baker el al. , Biophys. J. (1991) 60:1445-1456; Hagensee et al, J. Virol. (1994) 68:4503-4505.
  • VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding.
  • cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation and images recorded under appropriate exposure conditions. Additional methods of VLP purification include but are not limited to chromatographic techniques such as affinity, ion exchange, size exclusion, and reverse-phase procedures.
  • this disclosure further provides a cell or cell line comprising a nucleic acid molecule described above.
  • the cell or cell line further comprises a second nucleic acid molecule comprising a coding sequence of a VSV-G protein or a variant/fragment thereof.
  • the VSV-G protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or comprises the amino acid sequence of SEQ ID NO: 2.
  • the cell or cell line further comprises a third nucleic acid molecule comprising a coding sequence of a Spike protein or a variant/fragment thereof.
  • the Spike protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or comprises the amino acid sequence of SEQ ID NO: 4.
  • a representative amino acid sequence of the Spike protein is provided below (Accession ID: NC_045512.2; SEQ ID NO: 4):
  • the method may include introducing a nucleic acid molecule described above to a host cell, such as an animal cell.
  • the method may further include culturing the cell in a cell culture medium to obtain the coronavirus replicon-harbored cell.
  • the method further comprises introducing to the cell a second nucleic acid comprising a coding sequence of the YSV-G protein or a variant/fragment thereof.
  • the method may further comprise introducing to the cell a third nucleic acid comprising a coding sequence of the Spike protein or a variant/fragment thereof.
  • the terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • the nucleic acid molecule is introduced into a host cell by an electroporation procedure or a biolistic procedure.
  • host cells can be genetically engineered (e.g, transduced or transformed or transfected) with, for example, a vector construct of this disclosure.
  • the vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the vector containing a polynucleotide sequence as described herein, e.g, a nucleic acid molecule comprising a modified coronavirus genome or RNA replicon, as well as, optionally, a selectable marker or reporter gene, can be employed to transform an appropriate host cell.
  • Suitable host cells can include, but are not limited to, algal cells, bacterial cells, heterokonts, fungal cells, chytrid cells, microfungi, microalgae, and animal cells.
  • the animal cells are invertebrate animal cells.
  • the vertebrate animal cells are mammalians cells.
  • Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.
  • the methods disclosed herein can be used with host cells from species that are natural hosts of coronaviruses, such as rodents, mice, fish, birds, and larger mammals such as humans, horses, pig, monkey, and apes, as well as invertebrates.
  • any animal species can be generally used and can be, for example, human, dog, bird, fish, horse, pig, primate, mouse, cattle, swine, sheep, rabbit, cat, goat, donkey, hamster, or buffalo.
  • suitable bird species include chicken, duck, goose, turkey, ostrich, emu, swan, peafowl, pheasant, partridge, and guinea fowl.
  • the fish species is a salmon species.
  • Primary mammalian cells and continuous/immortalized cells types are also suitable.
  • suitable animal host cells include, but not limited to, pulmonary equine artery endothelial cell, equine dermis cell, baby hamster kidney (BHK) cell, rabbit kidney cell, mouse muscle cell, mouse connective tissue cell, human cervix cell, human epidermoid larynx cell, Chinese hamster ovary cell (CHO), human HEK-293 cell, mouse 3T3 cell, Vero cell, Madin- Darby Canine Kidney Epithelial Cell (MDCK), primary chicken fibroblast cell, a HuT78 cell, a Huh-7 cell, A549 lung cell, HeLa cell, PER.C6® cell, WI-38 cell, MRC-5 cell, FRhL-2, and CEM T-cell.
  • pulmonary equine artery endothelial cell equine dermis cell
  • BHK baby hamster kidney
  • the host cell is a baby hamster kidney cell. In some embodiments, the baby hamster kidney cell is a BHK-21 cell. In some embodiments, the host cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the host cell is a Huh-7.5 cell. In some embodiments, the cell is a lung organoid.
  • composition comprising a nucleic acid molecule or a cell or cell line, as described above, and a pharmaceutically acceptable carrier.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subj ect such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject.
  • materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer
  • “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
  • this disclosure provides a kit comprising a nucleic acid molecule, a cell or cell line, or the composition, as described above.
  • the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative.
  • the composition can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile.
  • the liquid solution preferably is an aqueous solution.
  • reconstitution generally is by the addition of a suitable solvent and acidulant.
  • the acidulant and solvent e.g., an aprotic solvent, sterile water, or a buffer
  • the kit may further include informational materials.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about the production of the composition, concentration, date of expiration, batch or production site information, and so forth.
  • RNA replicons can be used as a low-containment platform for molecular virology studies and drug development screening. Accordingly, this disclosure further provides a method for screening for antiviral agents for a coronavirus.
  • the method comprises: (i) contacting a cell or cell line described above with a candidate agent (e.g., test compound); and (ii) determining an increase or decrease in replication or activity of the coronavirus virus replicon relative to a control cell or cell line harboring the same replicon, wherein the control cell or cell line has not been contacted with the candidate agent.
  • a candidate agent e.g., test compound
  • the coronavirus is SARS-CoV or SARS-CoV-2.
  • the step of determining comprises determining a level of production of a coronavirus protein or a coronavirus RNA transcript.
  • antiviral agents can be an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate.
  • test compound examples include small organic or inorganic molecules, proteins, peptides, peptidomimetics, polysaccharides, nucleic acids, nucleic acid analogues and derivatives, or peptoids.
  • Candidate compounds to be screened e.g., proteins, peptides, peptidomimetics, peptoids, antibodies, small molecules, or other drugs
  • proteins, peptides, peptidomimetics, peptoids, antibodies, small molecules, or other drugs can be isolated from naturally occurring substances or obtained using any of the numerous approaches in combinatorial library methods known in the art.
  • Such libraries include: peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that is resistant to enzymatic degradation); spatially addressable parallel solid phase or solution phase libraries; synthetic libraries obtained by deconvolution or affinity chromatography selection; and the “one-bead one-compound” libraries. See, e.g., Zuckermann et al. 1994, J. Med. Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145.
  • a candidate compound/composition identified by the evaluation method can be further tested to confirm its therapeutic effect or modified to optimize its effect and limit any side effects, and then formulated as a therapeutic agent.
  • Therapeutic agents thus identified can be used in a therapeutic protocol to treat coronavirus infection.
  • the term “determining” means methods that include detecting the presence or absence or a level of marker(s) (e.g ., a coronavirus protein or a coronavirus RNA transcript) in the sample, quantifying the amount of marker(s) in the sample, and/or qualifying the type of biomarker. Measuring can be accomplished by methods known in the art and those further described herein.
  • the level of the one or more markers in a sample obtained from a subject may be determined by any of a wide variety of well-known techniques and methods, which transform a marker within the sample into a moiety that can be detected and quantified.
  • Non-limiting examples of such methods include analyzing the sample using immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods, immunoblotting, Western blotting, Northern blotting, electron microscopy, mass spectrometry, e.g., MALDI-TOF and SELDI-TOF, immunoprecipitations, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assays (ELISAs), e.g., amplified ELISA, quantitative blood-based assays, e.g., serum ELISA, quantitative urine-based assays, flow cytometry, Southern hybridizations, array analysis, and the like, and combinations or sub
  • the level of a marker in a sample can be determined by detecting a transcribed polynucleotide or portion thereof, e.g., mRNA, or cDNA, of a marker gene.
  • RNA may be extracted from cells using RNA extraction techniques, including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland).
  • Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton el al, (1984) Nuc. Acids Res. 12:7035-56), Northern blotting, in situ hybridization, and microarray analysis.
  • More than one antiviral agents can be tested at the same time for their ability to modulate the expression and/or activity of a marker in a screening assay.
  • screening assay refers to assays that test the ability of a plurality of compounds to influence the readout of choice rather than to tests that test the ability of one compound to influence a readout.
  • the assays may identify compounds not previously known to have the effect that is being screened for.
  • high throughput screening HTS can be used to assay for the activity of a compound.
  • Gene is used broadly to refer to any segment of a nucleic acid molecule that encodes a protein or that can be transcribed into a functional RNA.
  • Genes may include sequences that are transcribed but are not part of a final, mature, and/or functional RNA transcript, and genes that encode proteins may further comprise sequences that are transcribed but not translated, for example, 5’ untranslated regions, 3’ untranslated regions, introns, etc.
  • genes may optionally further comprise regulatory sequences required for their expression, and such sequences may be, for example, sequences that are not transcribed or translated.
  • Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
  • a “coding sequence” or a sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • nucleic acid molecule and “polynucleotide” are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules can have any three-dimensional structure. A nucleic acid molecule can be double- stranded or single-stranded (e.g., a sense strand or an antisense strand).
  • Non-limiting examples of nucleic acid molecules include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, tracrRNAs, crRNAs, guide RNAs, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes, and nucleic acid primers.
  • a nucleic acid molecule may contain unconventional or modified nucleotides.
  • the nomenclature for nucleotide bases as set forth in 37 CFR ⁇ 1.822 is used herein.
  • Nucleic acid molecules can be nucleic acid molecules of any length, including but not limited to, nucleic acid molecules that are between about 3 Kb and about 50 Kb, for example, between about 3 Kb and about 40 Kb, between about 3 Kb and about 40 Kb, between about 3 Kb and about 30 Kb, between about 3 Kb and about 20 Kb, between 5 Kb and about 40 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.
  • the nucleic acid molecules can also be, for example, more than 50 kb.
  • polynucleotides of the present disclosure can be “biologically active” with respect to either a stmctural attribute, such as the capacity of a nucleic acid to hybridize to another nucleic acid, or the ability of a polynucleotide sequence to be recognized and bound by a transcription factor and/or a nucleic acid polymerase.
  • a stmctural attribute such as the capacity of a nucleic acid to hybridize to another nucleic acid, or the ability of a polynucleotide sequence to be recognized and bound by a transcription factor and/or a nucleic acid polymerase.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), and translation termination sequences, and/or sequence elements controlling an open chromatin structure see e.g., McCaughan etal. (1995) PNAS USA 92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355.
  • the term “construct” is intended to mean any recombinant nucleic acid molecule such as an expression cassette, plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single- stranded or double- stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g. operably linked.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as “gene products.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • cells include the primary subject cells and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment); however, such altered progeny are included in these terms, so long as the progeny retain the same functionality as that of the originally transformed cell.
  • variant refers to a first molecule that is related to a second molecule (also termed a “parent” molecule).
  • the variant molecule can be derived from, isolated from, based on or homologous to the parent molecule.
  • a “functional variant” of a protein as used herein refers to a variant of such protein that retains at least partially the activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or replacement mutants), including polymorphs, etc. Also included within functional variants are fusion products of such protein with another, usually unrelated, nucleic acid, protein, polypeptide, or peptide. Functional variants may be naturally occurring or may be man-made.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the Cas protein with one or more conservative modifications may retain the desired functional properties, which can be tested using the functional assays known in the art.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • a homolog has a greater than 60% sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.
  • substantially identity as applied to polypeptides, means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75% sequence identity.
  • a peptide or polypeptide “fragment” as used herein refers to a less than full-length peptide, polypeptide or protein.
  • a peptide or polypeptide fragment can have at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids in length, or single unit lengths thereof.
  • fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length.
  • peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less than about 250 amino acids in length.
  • variants and homologs may have sequences with at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequences of transgenes described herein.
  • disease as used herein is intended to be generally synonymous and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
  • the term “modulate” is meant to refer to any change in biological state, i.e., increasing, decreasing, and the like.
  • the terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
  • “reduced,” “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example, a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • the terms “increased,” “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,” “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • a biological macromolecule such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide
  • an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • the activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • therapeutic agent refers to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • sample can be a sample of, serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells (e.g., antibody-producing cells) or tissue.
  • sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
  • sample and biological sample as used herein generally refer to a biological material being tested for and/or suspected of containing an analyte of interest such as antibodies.
  • the sample may be any tissue sample from the subject.
  • the sample may comprise protein from the subject.
  • inhibitor and “antagonize,” as used herein, mean to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely.
  • Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down-regulate a protein, a gene, and mRNA stability, expression, function and activity, e.g., antagonists.
  • in vitro' refers to events that occur in an artificial environment, e.g, in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • in vivo refers to events that occur within a multi-cellular organism, such as a non-human animal.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.
  • each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.
  • Table 1 contains the primers used for fragment cloning.
  • Table 1 List of primers for SARS-CoV-2 replicon construction - Organized by PCR reaction, final fragments for subcloning and yeast transformation are highlighted in bold, all others are intermediate overlap PCR templates.
  • DNA fragments 2-6 and 8 were the same as described in Thao et al. (PMID: 32365353). The rest of the fragments were PCR-amplified from SARS-CoV-2 clone 3.1 (Thao et al. PMID: 32365353), except fragment 7, which was amplified from cDNA obtained by RT-PCR of viral RNA extracted from isolate USA-WA1/2020 (BEI #NR-5281), grown in Vero-E6 cells. RNA was extracted from cells using Trizol (ThermoFisher #15596026) and RNeasy mini kit (Qiagen #74104), and cDNA was prepared using SuperscriptTM IV First-Strand Synthesis System with random primers (Thermo #18091050).
  • PCR amplification reactions were performed using KOD Xtreme Hot Start DNA Polymerase (EMD Millipore #71975). Accessory sequences, such as Neon Green, Glue, NeoR, were amplified from plasmids or purchased as synthetic DNA (IDT). PCR amplicons of fragments
  • SARS-CoV-2 nspl and nspl2 were done using the two-step PCR method on fragments 2 and 7, respectively, using the primers listed in Table 1.
  • Pol(-) mutant was created by mutating SARS-CoV-2 RdRp (nspl2) D760, D761 catalytic residues to N760, N761 (SDD/SNN) (41).
  • Nspl mutant that does not bind the 40S ribosome was created by mutating K164A/H165A (28, 29). The mutated fragments were used for replicon assembly, as detailed below.
  • DNA fragments for assembly were prepared by restriction digestion or PCR as detailed in Table 2, and agarose gel was extracted. Table 2 Preparation of fragments for yeast transformation associated recombination
  • Yeast assembly was performed according to the protocol in Thao eial., 2020 (42). Briefly, 50-100 ng of each fragment was mixed in an equimolar ratio and transformed into Saccharomyces cerevisiae (S. cerevisiae) strain VL6-48N. Transformed yeast was grown for 2-3 days on selective -HIS plates at 30°C. 4-10 colonies from each plate were picked, re-streaked on a new plate, and grown for 2 days at 30°C.
  • the plasmid prep was digested with BamHI-HF enzyme (NEB #R3136T), which does not have restriction sites in any of the pCCl-BAC-His3- replicon plasmids.
  • BamHI-HF enzyme NEB #R3136T
  • DNA was digested with Plasmid-SafeTM ATP -Dependent DNase (Lucigen #E3101K) for 24h and cleaned by extraction with Phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation.
  • Final DNA concentration was measured using Qbit dsDNA HS Assay (ThermoFisher #Q32851)
  • MPA Multiple displacement amplification
  • Amplified DNA was digested with Notl-HF enzyme (NEB #R3189S) and cleaned up with phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation.
  • Notl-HF enzyme NEB #R3189S
  • phenol-chloroform-isoamylalcohol SigmaAldrich #77617
  • Sindbis replicon RNA was in-vitro transcribed from a SinRep5-GFP plasmid linearized with Xhol (NEB) (6), using SP6 mMessage mMachine High Yield Capped RNA Transcription kit (ThermoFisher #AM1340) RNA transcripts were electroporated into Huh7.5 or BHK-21 cells using adapted protocols originally developed for launching HCV (43). Briefly, Huh7.5 or BHK-21 cells were trypsinized, washed twice with ice-cold phosphate-buffered saline (PBS) (Invitrogen), and resuspended at 1.5 x 10 7 cells/ml in PBS.
  • PBS ice-cold phosphate-buffered saline
  • SARS-CoV-2 replicon RNA and 2 pg of SARS- CoV2 N mRNA were mixed with 0.4 ml of cell suspension in a 2-mm cuvette (BTX #45-0125) and immediately pulsed using a BTX ElectroSquare Porator ECM 830 (860V, 99 ps, five pulses). Electroporated cells were incubated at room temperature for 10 min prior to resuspension in plating media.
  • BHK-21 cells ATCC CCL-10, M. auratus
  • MEM Minimum Essential Medium
  • Calu-3 cells ATCC® HTB-55TM, H. sapiens ; sex: male
  • EMEM Eagle’s Minimum Essential Medium
  • TMEM41B-KO and dox-inducible TMEM4 IB-reconstituted Huh-7.5 cells were previously described (45). TMEM41B expression was induced by Doxy cy cline at least 24h before electroporation.
  • Huh7.5 cells containing repSARS-CoV-2 Glue, minirepSARS-CoV-2 Glue, or repSARS- CoV-2 Glue pol- replicons were seeded onto 12-well plates in triplicate at 1 x 10 5 cells/well and treated with lOOnM remdesivir or DMSO vehicle. After incubating for 24 or 48 hours at 37 °C, supernatants were aspirated, cells were washed three times with PBS and subsequently lysed in 250 m ⁇ Tri-reagent (Zymo, cat. #R2050) per well. RNA was extracted using the Direct-zol RNA Miniprep Plus kit (Zymo Research, cat.
  • RPS11 forward: 5’- GCCGAGACTATCTGCACTAC-3’ (SEQ ID NO: 147) and reverse: 5’- ATGTCCAGCCTCAGAACTTC-3’ (SEQ ID NO: 148)
  • SARS-CoV-2 subgenomic N Leader forward: 5’-GTTTATACCTTCCCAGGTAACAAACC-3’ (SEQ ID NO: 149) and N reverse: 5’-GTAGAAATACCATCTTGGACTGAGATC-3’ (SEQ ID NO: 150)).
  • SARS-CoV-2 primers targeting genomic N are from Chu et a!., 2020.
  • the following PCR conditions were used: 50 °C for 2 min and 95 °C for 2 min (initial denaturation); 45 cycles 95 °C for 1 sec, 60 °C for 30 sec (PCR); followed by 95 °C for 15 sec, 65 °C for 10 sec, a slow increase to 95 °C (0.07 °C/sec) for a melt curve.
  • the data were analyzed by melt curve analysis for product specificity as well as AACT analysis for fold changes (after normalization to housekeeping genes) and graphed using Prism 8 (GraphPad).
  • Fluorescent and brightfield images were taken with Nikon Eclipse TE300 fluorescent microscope at xlO magnification, using NIS-Elements 4.10.01 software (Nikon).
  • Flow cytometry was performed on a minimum of 10,000 single cells/sample using LSRII Flow cytometer (BD Biosciences). Data analysis was done using FloJo software (BD Biosciences).
  • AM580 was purchased from Cayman Chemical (#15261), Remdesivir and Masitinib were purchased from MedChemExpress (#HY- 104077 and #HY- 10209 respectively), 27- hydroxycholesterol (27HC) was purchased from Sigma Aldrich (#SML2042), Human IFN Alpha A (Alpha 2a) and Human IFN Beta (la) were purchased from Pbl assay science (#11100-1 and #11410-2 respectively).
  • BHK-21 cells were transfected with VSV-G or control plasmid, using Lipofectamine 3000 (ThermoFisher #L3000001), using a reverse-transfection protocol. 24h post transfection, 6 million cells were electroporated with 5ug replicon and 2ug N protein mRNA as detailed above. Each three electroporation reactions were combined into a T175 flask. Medium was replaced after a few hours overnight to remove free-floating RNA and dead cells.
  • nspl-nspl6 non-structural proteins
  • S structural proteins-spike
  • M membrane
  • E envelope
  • N nucleocapsid-and eight accessory proteins (3a, 3b, 6, 7a, 7b, 8b, 9b and 14) expressed from sub-genomic RNAs (FIG. 1A) (77).
  • a modulatory design was adopted to assemble two versions: a “minimal” replicon consisting of viral 5’ and 3’UTRs, Orfla/b, and N encoding regions, and a “full” replicon consisting of all viral proteins with the exception of spike (S).
  • the spike transcription-regulating sequence TRS was used to drive expression of a gene cassette consisting of neomycin-resistance (NeoR) and a reporter gene (nuclear-localized NeonGreen, or secreted Gaussia luciferase) separated by a T2A self-cleaving sequence (FIG. 1A).
  • Both versions contain an upstream T7 promoter for in vitro transcription at the 5’ end and a self-cleaving HDV ribozyme at the 3’ end, which cleaves immediately after an encoded polyA sequence.
  • RNA virus reverse genetics system in yeast was utilized (18). This system leverages transformation-associated recombination (TAR) to efficiently and accurately assemble numerous, large overlapping DNA fragments (19). After transforming yeast with equimolar ratios of replicon fragments and confirming proper assembly with multiplex PCR, restriction digests of the resulting DNA were performed to determine plasmid integrity. Using a spike deleted NeonGreen reporter SARS-CoV-2 replicon for optimization, yeast-derived plasmids were contaminated with genomic DNA and did not reveal the expected Ndel digest pattern (FIG. IB). To circumvent this, plasmid safe (PS) DNAse treatment was used to remove contaminating yeast genomic DNA.
  • PS plasmid safe

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Abstract

This disclosure provides RNA replicons (e.g., SARS-CoV-2 RNA replicons) that can be trans-packaged for single-cycle delivery into a wide array of cell types and recapitulate all major enzymatic activities of intracellular viral replication. As a low-containment platform, the disclosed RNA replicons are broadly amenable to molecular virology studies and drug development screening efforts.

Description

CORONA VIRUS REPLICON DELIVERY PARTICLES AND METHODS OF USE
THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63/133,940, filed January 5, 2021. This application also claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63/187,233, filed May 11, 2021. The foregoing applications are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
The present invention relates to coronavirus reporter replicons and method of use thereof and more specifically to trans-packaged coronavirus reporter replicons and method of use thereof.
BACKGROUND OF THE INVENTION
SARS-CoV-2, the causative agent of COVID-19, is a member of the Coronaviridae family of positive sense ssRNA viruses. Similar to SARS-CoV and Middle East respiratory syndrome- related coronavirus (MERS-CoV), the pathogenicity of SARS-CoV-2 necessitates that molecular virology studies of infectious virus can only occur in high containment biological safety level 3 (BSL3) laboratory settings. While studies of viral components in BSL2 settings have been invaluable to establish initial lists of putative host factors and to refine viral enzymatic mechanisms, non-infectious systems permitting the study of intracellular replication have been lacking. Along similar lines, recent efforts to develop chimeric and pseudotyped viruses using either rhabdovirus- or lentivirus-based backbones with SARS-CoV-2 Spike have enabled rapid screening of neutralizing antibodies directed against Spike in BSL2 settings, though BSL3 confirmation with live-virus is typically required. There remains an urgent need for non-infectious platforms that permit the study of core SARS-CoV-2 enzymatic processes in a replication context.
Self-replicating RNAs, known as replicons, have provided an important model system to study numerous aspects of RNA virus life cycles. Replicons are typically constructed using reverse genetics systems to replace one or more viral structural proteins with selectable or reporter genes. Upon transfection or electroporation of viral replicon RNA into cells, the replicon RNA is translated to produce viral enzymes and cofactors necessary to establish RNA replication factories, with reporter genes providing convenient readouts for replicon viability and for the selection of non-cytopathic variants. Crucially, as key structural components of the virion are missing, RNA replication, translation, and functions of viral gene products proceed without producing infectious virus. Such systems have been invaluable as molecular virology and high-throughput compound screening and drug development platforms, most notably with hepatitis C virus.
There exists a pressing need for flexible vaccine platforms for cancer and infectious diseases, including for SARS-CoV-2.
SUMMARY OF THE INVENTION
This disclosure addresses the need mentioned above in a number of aspects by providing RNA replicons (e.g., SARS-CoV-2 RNA replicons) that can be trans-packaged for single-cycle delivery (termed “trans-packaged replicons (TPRs)”) into a wide array of cell types and recapitulate all major enzymatic activities of intracellular viral replication. As used herein, the terms “trans-packaged replicons (TPRs)” and “replicon delivery particles (RDPs)” are used interchangeably.
In one aspect, this disclosure provides a nucleic acid molecule comprising or encoding a coronavirus replicon. The replicon comprises: (i) a genomic or subgenomic nucleotide sequence of a coronavirus, wherein the nucleotide sequence comprises at least one of the coding sequences of a membrane (M) protein of the coronavirus and an envelope (E) protein of the coronavirus and wherein the coding sequence of a spike (S) protein of the coronavirus is inactivated or deleted; and (ii) a second nucleotide sequence encoding a selectable marker suitable for selection, wherein the selectable marker is under the control of the RNA virus replication machinery.
In some embodiments, the replicon lacks the Spike-coding sequence, which can be replaced by a reporter gene cassette to monitor replicon activity. Examples include Trans- packaged Spike deleted Nspl mutant replicon (TPR AS+) in which Nspl is modified to inhibit its activity, Trans-packaged Spike deleted Minus Accessory replicon (TPR AAcc) which lacks all accessory genes, Trans-packaged Spike deleted Minus SEM replicon (TPR ASEM) which lacks all structural genes, and Trans-packaged Spike deleted minimal replicon (miniTPR) which lacks all accessory and structural genes. In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
In some embodiments, the coding sequence of the spike protein or a portion thereof is replaced with the second nucleotide sequence. In some embodiments, the selectable marker is a gene that confers resistance to an antibiotic.
In some embodiments, the nucleic acid molecule further comprises a reporter gene. In some embodiments, the reporter gene is selected from the group consisting of NeonGreen, gaussia luciferase (Glue), mScarlet, green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced
GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Venus, mOrange, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, t-dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein, a Phycobiliprotein, and a biologically active variant or fragment of thereof.
In some embodiments, the reporter gene is operatively linked to a spike transcription regulating sequence (TRS). In some embodiments, the reporter gene is operatively linked to the TRS through a T2A self-cleaving sequence.
In some embodiments, the nucleic acid molecule has at least 80% sequence identity to SEQ ID NO: 1 or 3, or comprises the nucleic acid sequence of SEQ ID NO: 1 or 3.
In another aspect, this disclosure also provides a virus particle or a virus-like particle comprising a nucleic acid molecule described above. In some embodiments, the virus particle or virus-like particle comprises a vesicular stomatitis virus G (VSV-G) protein or a variant/fragment thereof. In another aspect, this disclosure additionally provides a cell or cell line comprising a nucleic acid molecule described above. In some embodiments, the cell or cell line further comprises a second nucleic acid molecule comprising a coding sequence of a VSV-G protein or a variant/fragment thereof. In some embodiments, the VSV-G protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or comprises the amino acid sequence of SEQ ID NO: 2. VSV-G protein sequence (SEQ ID NO: 2):
MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQ VKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTK QGTWLNPGFPPQSCGYAFVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICP TVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKA CKMQ Y CKHW GVRLP SGVWFEMADKDLF AAARFPECPEGS SIS AP SQT S VD V SLIQD VE RILDY SLCQETW SKIRAGLPISPVDLS YLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPI L SRMVGMIS GTTTEREL WDD W AP YED VEIGPN GVLRT S S GYKFPL YMIGHGMLD SDLH LSSKAQWEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLII GLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK
In some embodiments, the cell or cell line further comprises a third nucleic acid molecule comprising a coding sequence of a Spike protein or a variant/fragment thereof In some embodiments, the Spike protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or comprises the amino acid sequence of SEQ ID NO: 4.
In some embodiments, the cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the cell is a Huh-7.5 cell or a BHK-21 cell. In some embodiments, the cell is a lung organoid.
Also within the scope of this disclosure is a composition comprising a nucleic acid molecule or a cell or cell line, as described above, and a pharmaceutically acceptable carrier.
In another aspect, this disclosure provides a kit comprising a nucleic acid molecule, a cell or cell line, or a composition, as described above.
In another aspect, this disclosure further provides a method of preparing a coronavirus replicon-harbored cell. The method comprises: (i) introducing a nucleic acid molecule described above to a cell; and (ii) culturing the cell in a cell culture medium to produce the coronavirus replicon-harbored cell. The cell can contain a second nucleic acid comprising a coding sequence of the VSV-G protein or a variant/fragment thereof. Accordingly, in some embodiments, the method may further comprise introducing to the cell a second nucleic acid comprising a coding sequence of the VSV-G protein or a variant/fragment thereof. In some embodiments, the method may further comprise introducing to the cell a third nucleic acid comprising a coding sequence of the Spike protein or a variant/fragment thereof. In some embodiments, the cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the cell is a Huh-7.5 cell.
In yet another aspect, this disclosure further provides a method for screening for antiviral agents for a coronavirus. The method comprises: (i) contacting a cell or cell line described above with a candidate agent; and (ii) determining an increase or decrease in replication or activity of the coronavirus virus replicon relative to a control cell or cell line harboring the same replicon, wherein the control cell or cell line has not been contacted with the candidate agent. In some embodiments, the coronavirus is SARS-CoV or SARS-CoV-2.
In some embodiments, the step of determining comprises determining a level of production of a coronavirus protein or a coronavirus RNA transcript.
In some embodiments, the candidate agent comprises an organic compound or an antisense nucleic acid.
The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, IB, 1C, ID, IE, IF, and 1G are a set of diagrams showing SARS-CoV-2 replicon design and launch optimization. FIG. 1A is a schematic of a modular SARS-CoV-2 replicon design. Fragments from (18) are shown in orange, and fragments harboring mutations in nspl or the RdRP (nspl2) are shown in red. Fragments to encode a spike-replaced reporter gene cassette, accessory proteins, and flanking regions are shown in purple, green, or blue, respectively. FIG. IB shows agarose gel of replicon DNA recovered from yeast or bacteria, before or after amplification with phi29 DNA polymerase. Yeast derived plasmids were further treated with Plasmid-Safe (PS) DNAse as indicated. Digestion with Ndel was used to verify plasmid integrity based on the expected banding pattern depicted at right. FIG. 1C shows agarose gel of T7 RNA transcription reactions from DNA plasmids shown in FIG. IB. Arrow highlights the expected size of full-length RNA. Asterisk denotes a non-specific band. FIG. ID shows percent of NeonGreen SARS-CoV-2 replicon positive BHK-21 cells from non-amplified T7 transcription reactions measured by flow cytometry. Inset shows representative NeonGreen (NG) and bright-field (BF) images. N = 3, error bars = SEM. FIG. IE shows percent of NeonGreen SARS-CoV-2 replicon positive BHK-21 cells from phi29 amplified T7 transcription reactions measured by flow cytometry. Inset shows representative NeonGreen (NG) and bright-field (BF) images. N = 3, error bars = SEM. FIG. IF shows a flowchart diagram of optimized RNA production for SARS-CoV-2 replicons. Viral fragments and reporter transgenes are cloned and assembled in yeast. Yeast derived plasmids can either be propagated in bacteria or in yeast, in which case they are treated with plasmid-safe DNAse to remove DNA contaminants. Subsequent phi29 MDA amplification ensures full-length DNA template availability for T7 transcription. FIG. 1G shows percent of NeonGreen SARS-CoV-2 replicon positive BHK-21 cells from phi29 amplified T7 transcription reactions using various capping strategies, measured by flow cytometry. N = 3, error bars = SEM.
FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 21, and 2J are a set of diagrams showing that SARS- CoV-2 replicons are sensitive to antiviral compounds, required host factor loss, and viral mutant phenotypes. FIG. 2A shows cumulative timecourse measurements of gaussia luciferase (Glue) in the supernatants of Huh-7.5 cells electroporated with the Glue replicon and seeded with 100 nM remdesivir (Rem) or vehicle. A polymerase deficient (pol-) replicon was used as negative control. Dashed line indicates the limit of detection. Error bars represent standard deviation of 3 biological repeats. FIG. 2B shows qRT-PCR measurements for subgenomic N RNA in Huh-7.5 cells harboring Glue replicons electroporated with the Glue replicon and seeded with lOOnM remdesivir (Rem) or vehicle. A polymerase deficient (pol-) replicon was used for normalization (dashed line). Error bars represent standard deviation of 3 biological repeats. FIG. 2C shows that Huh-7.5 Cells were electroporated with the Glue replicon and seeded in 96-wells plates on a dose-curve of remdesivir. 24h post electroporation, GLuc signal was measured in sup (black circles), and cell viability was measured by cell-titer Glo assay (empty circles). Data was normalized to vehicle (DMSO)-treated cells. Error bars represent standard deviation of 5 biological repeats. FIG. 2D is the same as FIG. 2C except that the cells were treated with Masitinib. Error bars represent standard deviation of 4 biological repeats. FIG. 2E is the same as FIG. 2C except that the cells were treated with 27-hydroxycholesterol (27HC). Error bars represent standard deviation of 3 biological repeats. FIG. 2F shows that WT or TMEM41B KO cells were electroporated with the GLuc replicon and seeded with lOOnM Remdesivir or vehicle. GLuc was measured 24h post electroporation. Error bars represent standard deviation of 4 biological repeats. FIG. 2G shows that cells in F were electroporated with SinRep-GFP Sindbis virus replicon RNA and seeded in 6-wells plates. Percent of GFP-positive cells was determined by flow cytometry 24h post electroporation. Error bars represent standard deviation from three biological replicates. FIG. 2H is the same as FIG. 2F except that TMEM41B KO and TMEM41B KO cells reconstituted with dox-inducible TMEM41B were used. Both cell lines were treated with 2 pg/ml doxycycline 24h before electroporation. FIG. 21 shows that Huh7.5 cells were electroporated with either WT or NSP1 K164A/H165A mutant (NSPl '). Electroporated cells were seeded with lOOnM Remdesivir or vehicle, and GLuc levels on the supernatant were measured at the indicated time points. Error bars represent standard deviation of 4 biological repeats. FIG. 2J is the same as FIG. 21 except that cell viability was measured at each time point by cell-titer Glo assay. Mock-electroporated cells were used as control for normal post-electroporation cell viability.
FIGS. 3A, 3B, 3C, 3D, 3E, and 3F are a set of diagrams showing kinetics and drug susceptibility of different replicons. FIG. 3 A shows that Huh7.5 cells were electroporated with WT or NSP- replicons and seeded on a dose-curve of Interferon alpha (IFNa). GLuc activity (closed circles) and cell viability (open circles) were measured 24 hours post electroporation. FIG. 3B is the same as FIG. 3A except that cells were treated with interferon beta (IFNp) FIG. 3C is the same as FIG. 3A except that cells were treated with Remdesivir. FIG. 3D is a schematic of the full replicon, containing most structural proteins excluding Spike and all the accessory proteins, and minireplicons, which are devoid of all accessory and structural proteins and encode only for the replicase and N genes. FIG. 3E shows kinetics of GLuc expression of the full and mini replicons. Huh7.5 cells were electroporated with the full or mini replicons and seeded with 100 nM Remdesivir (open circles) or vehicle (closed circles). GLuc activity in the supernatant was measured at the indicated time points. FIG. 3F is the same as FIG. 3E, except that cell viability was measured by cell titer Glo assay and normalized to day 1 post electroporation. Mock- electroporated cells were used as control for normal post-electroporation cell viability. FIGS. 4A, 4B, 4C, and 4D are a set of diagrams showing replicon delivery by transpackaging with VSV-G. FIG. 4 A shows an experimental scheme describing the trans-packaging of replicons with VSV-G. Briefly, BHK-21 cells were transfected with VGV-G, and the next day electroporated with the full SARS-CoV2 replicon. After 48h, the supernatant of the producer cells was collected and concentrated. Multiple cell types can be transduced with the resulting trans- packaged replicons (TRPs). FIG. 4B shows that indicated cells were seeded in 96-well plates and transduced with TPR-NeonGreen, produced as described in FIG. 4A. Brightfield and fluorescent images were taken at xlO magnification 24h after transduction. FIG. 4C shows that Huh7.5 cells were transduced with concentrated or 1:10 diluted TPR-NeonGreen, and percent of reporter positive cells was measured by flow cytometry. As negative controls, cells were transduced with similarly collected supernatants from cells that were electroporated with the minireplicon instead, or where VSV-G was replaced by a control plasmid. FIG. 4D shows that normal human bronchial epithelial cells (NHBE), normal human lung fibroblasts (NHLF), and A549 cells were pre-treated with lOOnM of Remdesivir or lOOpM of IFNa, and the next day transduced with TPR-GLuc. GLuc activity in the sup was measured 24h after transduction. Error bars represent standard deviation of 3 biological replicates. FIG. 5E shows that TPRs are single-cycle infectious virions. Huh7.5 cells were transduced with IOOmI of TPRs per 2ml in a 6-well format (P0 supernatant). After lhr, inoculum was saved and concentrated with PEG (P0’ supernatant). Cells were washed 4x in PBS and incubated with media for 24hr at 37C. The resulting PI supernatant was concentrated with PEG. Naive Huh7. cells were transduced with P0’ or PI supernatants, washed after lhr, and incubated for 24hrs at 37C followed by Glue measurement. Schema depicted at left, results at right for two experiments. Dashed line indicates signal from mock transduced cells.
FIGS. 5A, 5B, and 5C are a set of photographs and diagrams showing replicon assembly and RNA validation (Related to FIG. 1). FIG. 5A shows that six different replicons were assembled in yeast, and DNA from four colonies of each assembly were extracted and subjected to multiplex PCR with primer set #1 (see Table 1). Expected PCR product sizes are indicated on the right. FIG. 5B shows that RNA was in-vitro transcribed from lug of the indicated phi-29 amplified DNA templates, using T7 RiboMAX Express Large Scale RNA Production System (Promega) or HiScribe T7 High Yield RNA Synthesis kit (NEB). After DNAse-treatment and purification, lul (1:50 of the reaction) was loaded on an agarose gel. The black arrow indicates the full-length product. Percent of full-length RNA per lane is indicated at bottom. FIG. 5C shows the results of qRT-PCR measurements for N RNA in Huh-7.5 cells electroporated with the 5pg Glue replicon RNA plus 2pg N mRNA and seeded with lOOnM remdesivir (Rem) or vehicle. The signal from mock infected cells was used for normalization (dashed line). Error bars represent standard deviation of 3 biological repeats.
FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H are a set of diagrams showing susceptibility of the Spike-deleted and minireplicons to antiviral compounds (Related to FIG. 3). FIGS. 6A, 6B, 6C, and 6D show that Huh7.5 cells were electroporated with GLuc spike-deleted replicon and seeded in 96-wells plate on a dose-curve of Remdesivir (FIG. 6A), IFNa (FIG. 6B) IFNb (FIG. 6C) or AM580 (FIG. 6D). GLuc activity in the supernatant (closed circles) and cell viability (open circles) were measured 48h post electroporation. Error bars represent standard error from 3 biological repeats.
FIGS. 7A, 7B, 7C, 7D, 7E, and 7F are a set of diagrams showing trans-complementation of replicons with Spike yields single-cycle SARS-CoV-2. FIG. 7A shows a scheme to trans complement replicons with ectopically expressed Spike for single-cycle virion production. BHK- 21 cells are transfected with a Spike encoding plasmid, and 24 hours later electroporated with the full SARS-CoV2 replicon. Supernatant from these producer cells (P0) is collected and passaged onto recipient cells (PI) yielding reporter activity. A second round of passaging onto naive recipient cells (P2) fails to propagate the replicon. FIG. 7B shows a spike trans-packaged replicon consisting of the full Spike deleted replicon RNA alongside plasmid driven Spike expression. Nspl mutations relative to the WT sequence indicated. FIG. 7C shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer cells (P0) with supernatants concentrated and subsequently passaged twice onto Huh-7.5 ACE2/TMPRSS2 (Huh-7.5 AT) cells (PI and P2). Immunofluorescence images at 4X of the mNeongreen signal (green) and N antibody staining (magenta) are shown. Scale bar, lOOpm. FIG. 7D shows neongreen quantification per passage for the results in (FIG. 1C). Dashed lines denote the lower limit of quantification. N = 8, error bars = SD. FIGS. 7E and 7F show antibody neutralization assays in Huh-7.5 cells of Glue TPRs packaged with WT Spike (FIG. 7A) or the B.1.351 South African variant (FIG. 7B) in the presence of increasing concentrations of C144 and C135 neutralizing antibodies. N = 3, error bars = SEM.
FIGS. 8A, 8B, 8C, 8D, and 8E are a set of diagrams showing ectopic Spike expression and quantification. FIG. 8A shows that WT or Nspl- replicons were electroporated alone or in Spike transfected producer BHK-21 cells (P0) with supernatants subsequently passaged onto Huh-7.5 ACE2/TMPRSS2 (Huh-7.5 AT) cells (PI). Immunofluorescence images at 4X of the NeonGreen signal (green) and Spike C144 antibody staining (magenta) are shown. Scale bar, 100 pm. FIG. 8B shows Spike immunofluorescence quantification per passage for the results in (FIG. 8A). N = 2, error bars = SD. FIG. 8C shows N immunofluorescence quantification per passage for the results in Figure 4C. N = 2-6, error bars = SD. FIG. 8D shows immunofluorescence images N protein (magenta) in Huh-7.5 cells infected with SARS-CoV-2 (WA1/2020, left) or transduced with TPRs packaged with WT Spike (right). Images at 4X magnification, scale bar 500pm. FIG. 8E shows quantification of results in (FIG. 8D), measuring approximate per cell area for the N protein signal between virus or TPR positive cells.
FIGS. 9A, 9B, and 9C are a set of diagrams showing neutralization assays with TPRs recapitulate authentic SARS-CoV-2 antibody phenotypes. FIGS. 9A, 9B, and 9C show neutralization assays for SARS-CoV-2 TPRs or virus in the presence of increasing concentrations of antibodies. WT Spike with Cl 44 antibody (FIG. 9A), B.1.351 Spike with Cl 44 antibody (FIG. 9B), and B.1.351 with C135 antibody (FIG. 9C) are shown. N = 3, error bars = SEM.
DETAILED DESCRIPTION OF THE INVENTION
This disclosure provides RNA replicons (e.g, SARS-CoV-2 RNA replicons) that can be trans-packaged for single-cycle delivery into a wide array of cell types and recapitulate all major enzymatic activities of intracellular viral replication. As a low-containment platform, the disclosed RNA replicons are broadly amenable to molecular virology studies and drug development screening efforts.
A. NUCLEIC ACID MOLECULES AND RNA REPLICONS
In one aspect, this disclosure provides a nucleic acid molecule comprising or encoding a coronavirus replicon. In some embodiments, the replicon comprises: (i) a genomic nucleotide sequence of a coronavirus, wherein the nucleotide sequence comprises at least one of the coding sequences of a membrane (M) protein of the coronavirus and an envelope (E) protein of the coronavirus and wherein the coding sequence of a spike (S) protein of the coronavirus is inactivated or deleted; and (ii) a second nucleotide sequence encoding a selectable marker suitable for selection, wherein the selectable marker is under the control of the RNA virus replication machinery. In some embodiments, the selectable marker is a gene that confers resistance to an antibiotic.
In some embodiments, the coding sequence of the spike protein or a portion thereof is replaced with the second nucleotide sequence.
In some embodiments, the nucleic acid molecule further comprises a reporter gene. The reporter gene can be selected from the group consisting of NeonGreen, gaussia luciferase (Glue), green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Venus, mOrange, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t- HcRed, DsRed, DsRed2, t-dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein a Phycobiliprotein, and a biologically active variant or fragment of thereof.
In some embodiments, the reporter gene is operatively linked to a spike transcriptionregulating sequence (TRS). The reporter gene can be directly or indirectly linked to a linker or a cleavage site. For example, the reporter gene can be operatively linked to a TRS through a T2A self-cleaving sequence.
In some embodiments, the nucleic acid molecule has at least 80% sequence identity to SEQ ID NO: 1 or 3 (listed below), or comprises the nucleic acid sequence of SEQ ID NO: 1 or 3. pccl-bac-repsars-cov-2 neor-t2a-nlsneongreen.gb.xdna (“greenGB”), 35914 bp (SEQ ID NO:
1):
1 gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
61 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccacacagat
121 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt cagctgcgca actgttggga
181 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
241 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacTCATGC GTCCATAGTC
301 CCGTTCCGTA ATACGACTCA CTATAGatta aaggtttata ccttcccagg taacaaacca
361 accaactttc gatctcttgt agatctgttc tctaaacgaa ctttaaaatc tgtgtggctg
421 tcactcggct gcatgcttag tgcactcacg cagtataatt aataactaat tactgtcgtt
481 gacaggacac gagtaactcg tctatcttct gcaggctgct tacggtttcg tccgtgttgc
541 agccgatcat cagcacatct aggtttcgtc egggtgtgac cgaaaggtaa gatggagagc
601 cttgtccctg gtttcaacga gaaaacacac gtccaactca gtttgcctgt tttacaggtt
661 cgcgacgtgc tcgtacgtgg ctttggagac tccgtggagg aggtcttatc agaggcacgt
721 caacatctta aagatggcac ttgtggctta gtagaagttg aaaaaggcgt tttgcctcaa
781 cttgaacagc cctatgtgtt catcaaacgt tcggatgctc gaactgcacc tcatggtcat
841 gttatggttg agctggtagc agaactcgaa ggcattcagt acggtcgtag tggtgagaca
901 cttggtgtcc ttgtccctca tgtgggcgaa ataccagtgg cttaccgcaa ggttcttctt
961 cgtaagaacg gtaataaagg agctggtggc catagttacg gcgccgatct aaagtcattt 1021 gacttaggcg acgagcttgg cactgatcct tatgaagatt ttcaagaaaa ctggaacact
1081 aaacatagca gtggtgttac ccgtgaactc atgcgtgagc ttaacggagg ggcatacact
1141 cgctatgtcg ataacaactt ctgtggccct gatggctacc ctcttgagtg cattaaagac
1201 cttctagcac gtgctggtaa agcttcatgc actttgtccg aacaactgga ctttattgac
1261 actaagaggg gtgtatactg ctgccgtgaa catgagcatg aaattgcttg gtacacggaa
1321 cgttctgaaa agagctatga attgcagaca ccttttgaaa ttaaattggc aaagaaattt
1381 gacaccttca atggggaatg tccaaatttt gtatttccct taaattccat aatcaagact
1441 attcaaccaa gggttgaaaa gaaaaagctt gatggcttta tgggtagaat tcgatctgtc
1501 tatccagttg cgtcaccaaa tgaatgcaac caaatgtgcc tttcaactct catgaagtgt
1561 gatcattgtg gtgaaacttc atggcagacg ggcgattttg ttaaagccac ttgcgaattt
1621 tgtggcactg agaatttgac taaagaaggt gccactactt gtggttactt accccaaaat
1681 gctgttgtta aaatttattg tccagcatgt cacaattcag aagtaggacc tgagcatagt
1741 cttgccgaat accataatga atctggcttg aaaaccattc ttcgtaaggg tggtcgcact
1801 attgcctttg gaggctgtgt gttctcttat gttggttgcc ataacaagtg tgcctattgg
1861 gttccacgtg ctagcgctaa cataggttgt aaccatacag gtgttgttgg agaaggttcc
1921 gaaggtctta atgacaacct tcttgaaata ctccaaaaag agaaagtcaa catcaatatt
1981 gttggtgact ttaaacttaa tgaagagatc gccattattt tggcatcttt ttctgcttcc
2041 acaagtgctt ttgtggaaac tgtgaaaggt ttggattata aagcattcaa acaaattgtt
2101 gaatcctgtg gtaattttaa agttacaaaa ggaaaagcta aaaaaggtgc ctggaatatt
2161 ggtgaacaga aatcaatact gagtcctctt tatgcatttg catcagaggc tgctcgtgtt
2221 gtacgatcaa ttttctcccg cactcttgaa actgctcaaa attctgtgcg tgttttacag
2281 aaggccgcta taacaatact agatggaatt tcacagtatt cactgagact cattgatgct
2341 atgatgttca catctgattt ggctactaac aatctagttg taatggccta cattacaggt
2401 ggtgttgttc agttgacttc gcagtggcta actaacatct ttggcactgt ttatgaaaaa
2461 ctcaaacccg tccttgattg gcttgaagag aagtttaagg aaggtgtaga gtttcttaga
2521 gacggttggg aaattgttaa atttatctca acctgtgctt gtgaaattgt cggtggacaa
2581 attgtcacct gtgcaaagga aattaaggag agtgttcaga cattctttaa gcttgtaaat
2641 aaatttttgg ctttgtgtgc tgactctatc attattggtg gagctaaact taaagccttg
2701 aatttaggtg aaacatttgt cacgcactca aagggattgt acagaaagtg tgttaaatcc
2761 agagaagaaa ctggcctact catgcctcta aaagccccaa aagaaattat cttcttagag
2821 ggagaaacac ttcccacaga agtgttaaca gaggaagttg tcttgaaaac tggtgattta
2881 caaccattag aacaacctac tagtgaagct gttgaagctc cattggttgg tacaccagtt
2941 tgtattaacg ggcttatgtt gctcgaaatc aaagacacag aaaagtactg tgcccttgca
3001 cctaatatga tggtaacaaa caataccttc acactcaaag gcggtgcacc aacaaaggtt
3061 acttttggtg atgacactgt gatagaagtg caaggttaca agagtgtgaa tatcactttt
3121 gaacttgatg aaaggattga taaagtactt aatgagaagt gctctgccta tacagttgaa
3181 ctcggtacag aagtaaatga gttcgcctgt gttgtggcag atgctgtcat aaaaactttg
3241 caaccagtat ctgaattact tacaccactg ggcattgatt tagatgagtg gagtatggct
3301 acatactact tatttgatga gtctggtgag tttaaattgg cttcacatat gtattgttct
3361 ttctaccctc cagatgagga tgaagaagaa ggtgattgtg aagaagaaga gtttgagcca
3421 tcaactcaat atgagtatgg tactgaagat gattaccaag gtaaaccttt ggaatttggt
3481 gccacttctg ctgctcttca acctgaagaa gagcaagaag aagattggtt agatgatgat
3541 agtcaacaaa ctgttggtca acaagacggc agtgaggaca atcagacaac tactattcaa
3601 acaattgttg aggttcaacc tcaattagag atggaactta caccagttgt tcagactatt
3661 gaagtgaata gttttagtgg ttatttaaaa cttactgaca atgtatacat taaaaatgca
3721 gacattgtgg aagaagctaa aaaggtaaaa ccaacagtgg ttgttaatgc agccaatgtt
3781 taccttaaac atggaggagg tgttgcagga gccttaaata aggctactaa caatgccatg
3841 caagttgaat ctgatgatta catagctact aatggaccac ttaaagtggg tggtagttgt
3901 gttttaagcg gacacaatct tgctaaacac tgtcttcatg ttgtcggccc aaatgttaac
3961 aaaggtgaag acattcaact tcttaagagt gcttatgaaa attttaatca gcacgaagtt
4021 ctacttgcac cattattatc agctggtatt tttggtgctg accctataca ttctttaaga
4081 gtttgtgtag atactgttcg cacaaatgtc tacttagctg tctttgataa aaatctctat
4141 gacaaacttg tttcaagctt tttggaaatg aagagtgaaa agcaagttga acaaaagatc
4201 gctgagattc ctaaagagga agttaagcca tttataactg aaagtaaacc ttcagttgaa
4261 cagagaaaac aagatgataa gaaaatcaaa gcttgtgttg aagaagttac aacaactctg
4321 gaagaaacta agttcctcac agaaaacttg ttactttata ttgacattaa tggcaatctt
4381 catccagatt ctgccactct tgttagtgac attgacatca ctttcttaaa gaaagatgct 4441 ccatatatag tgggtgatgt tgttcaagag ggtgttttaa ctgctgtggt tatacctact 4501 aaaaaggctg gtggcactac tgaaatgcta gcgaaagctt tgagaaaagt gccaacagac 4561 aattatataa ccacttaccc gggtcagggt ttaaatggtt acactgtaga ggaggcaaag 4621 acagtgctta aaaagtgtaa aagtgccttt tacattctac catctattat ctctaatgag 4681 aagcaagaaa ttcttggaac tgtttcttgg aatttgcgag aaatgcttgc acatgcagaa 4741 gaaacacgca aattaatgcc tgtctgtgtg gaaactaaag ccatagtttc aactatacag 4801 cgtaaatata agggtattaa aatacaagag ggtgtggttg attatggtgc tagattttac 4861 ttttacacca gtaaaacaac tgtagcgtca cttatcaaca cacttaacga tctaaatgaa 4921 actcttgtta caatgccact tggctatgta acacatggct taaatttgga agaagctgct 4981 cggtatatga gatctctcaa agtgccagct acagtttctg tttcttcacc tgatgctgtt 5041 acagcgtata atggttatct tacttcttct tctaaaacac ctgaagaaca ttttattgaa 5101 accatctcac ttgctggttc ctataaagat tggtcctatt ctggacaatc tacacaacta 5161 ggtatagaat ttcttaagag aggtgataaa agtgtatatt acactagtaa tcctaccaca 5221 ttccacctag atggtgaagt tatcaccttt gacaatctta agacacttct ttctttgaga 5281 gaagtgagga ctattaaggt gtttacaaca gtagacaaca ttaacctcca cacgcaagtt 5341 gtggacatgt caatgacata tggacaacag tttggtccaa cttatttgga tggagctgat 5401 gttactaaaa taaaacctca taattcacat gaaggtaaaa cattttatgt tttacctaat 5461 gatgacactc tacgtgttga ggcttttgag tactaccaca caactgatcc tagttttctg 5521 ggtaggtaca tgtcagcatt aaatcacact aaaaagtgga aatacccaca agttaatggt 5581 ttaacttcta ttaaatgggc agataacaac tgttatcttg ccactgcatt gttaacactc 5641 caacaaatag agttgaagtt taatccacct gctctacaag atgcttatta cagagcaagg 5701 gctggtgaag ctgctaactt ttgtgcactt atcttagcct actgtaataa gacagtaggt 5761 gagttaggtg atgttagaga aacaatgagt tacttgtttc aacatgccaa tttagattct 5821 tgcaaaagag tcttgaacgt ggtgtgtaaa acttgtggac aacagcagac aacccttaag 5881 ggtgtagaag ctgttatgta catgggcaca ctttcttatg aacaatttaa gaaaggtgtt 5941 cagatacctt gtacgtgtgg taaacaagct acaaaatatc tagtacaaca ggagtcacct 6001 tttgttatga tgtcagcacc acctgctcag tatgaactta agcatggtac atttacttgt 6061 gctagtgagt acactggtaa ttaccagtgt ggtcactata aacatataac ttctaaagaa 6121 actttgtatt gcatagacgg tgctttactt acaaagtcct cagaatacaa aggtcctatt 6181 acggatgttt tctacaaaga aaacagttac acaacaacca taaaaccagt tacttataaa 6241 ttggatggtg ttgtttgtac agaaattgac cctaagttgg acaattatta taagaaagac 6301 aattcttatt tcacagagca accaattgat cttgtaccaa accaaccata tccaaacgca 6361 agcttcgata attttaagtt tgtatgtgat aatatcaaat ttgctgatga tttaaaccag 6421 ttaactggtt ataagaaacc tgcttcaaga gagcttaaag ttacattttt ccctgactta 6481 aatggtgatg tggtggctat tgattataaa cactacacac cctcttttaa gaaaggagct 6541 aaattgttac ataaacctat tgtttggcat gttaacaatg caactaataa agccacgtat 6601 aaaccaaata cctggtgtat acgttgtctt tggagcacaa aaccagttga aacatcaaat 6661 tcgtttgatg tactgaagtc aqaqqacqcq cagggaatgg ataatcttgc ctgcgaagat 6721 ctaaaaccag tctctgaaga agtagtggaa aatcctacca tacagaaaga cgttcttgag 6781 tgtaatgtga aaactaccga agttgtagga gacattatac ttaaaccagc aaataatagt 6841 ttaaaaatta cagaagaggt tggccacaca gatctaatgg ctgcttatgt agacaattct 6901 agtcttacta ttaagaaacc taatgaatta tctagagtat taggtttgaa aacccttgct 6961 actcatggtt tagctgctgt taatagtgtc ccttgggata ctatagctaa ttatgctaag 7021 ccttttctta acaaagttgt tagtacaact actaacatag ttacacggtg tttaaaccgt 7081 gtttgtacta attatatgcc ttatttcttt actttattgc tacaattgtg tacttttact 7141 agaagtacaa attctagaat taaagcatct atgccgacta ctatagcaaa gaatactgtt 7201 aagagtgtcg gtaaattttg tctagaggct tcatttaatt atttgaagtc acctaatttt 7261 tctaaactga t33^t3tt31 aatttggttt ttactattaa gtgtttgcct aggttcttta 7321 atctactcaa ccgctgcttt aggtgtttta atgtctaatt taggcatgcc ttcttactgt 7381 actggttaca gagaaggcta tttgaactct actaatgtca ctattgcaac ctactgtact 7441 ggttctatac cttgtagtgt ttgtcttagt ggtttagatt ctttagacac ctatccttct 7501 ttagaaacta tacaaattac catttcatct tttaaatggg atttaactgc ttttggctta 7561 gttgcagagt ggtttttggc atatattctt ttcactaggt ttttctatgt acttggattg 7621 gctgcaatca tgcaattgtt tttcagctat tttgcagtac attttattag taattcttgg 7681 cttatgtggt taataattaa tcttgtacaa atggccccga tttcagctat ggttagaatg 7741 tacatcttct ttgcatcatt ttattatgta tggaaaagtt atgtgcatgt tgtagacggt 7801 tgtaattcat caacttgtat gatgtgttac aaacgtaata gagcaacaag agtcgaatgt 7861 acaactattg ttaatggtgt tagaaggtcc ttttatgtct atgctaatgg aggtaaaggc
7921 ttttgcaaac tacacaattg gaattgtgtt aattgtgata cattctgtgc tggtagtaca
7981 tttattagtg atgaagttgc gagagacttg tcactacagt ttaaaagacc aataaatcct
8041 actgaccagt cttcttacat cgttgatagt gttacagtga agaatggttc catccatctt
8101 tactttgata aagctggtca aaagacttat gaaagacatt ctctctctca ttttgttaac
8161 ttagacaacc tgagagctaa taacactaaa ggttcattgc ctattaatgt tatagttttt
8221 gatggtaaat caaaatgtga agaatcatct gcaaaatcag cgtctgttta ctacagtcag
8281 cttatgtgtc aacctatact gttactagat caggcattag tgtctgatgt tggtgatagt
8341 gcggaagttg cagttaaaat gtttgatgct tacgttaata cgttttcatc aacttttaac
8401 gtaccaatgg aaaaactcaa aacactagtt gcaactgcag aagctgaact tgcaaagaat
8461 gtgtccttag acaatgtctt atctactttt atttcagcag ctcggcaagg gtttgttgat
8521 tcagatgtag aaactaaaga tgttgttgaa tgtcttaaat tgtcacatca atctgacata
8581 gaagttactg gcgatagttg taataactat atgctcacct ataacaaagt tgaaaacatg
8641 acaccccgtg accttggtgc ttgtattgac tgtagtgcgc gtcatattaa tgcgcaggta
8701 gcaaaaagtc acaacattgc tttgatatgg aacgttaaag atttcatgtc attgtctgaa
8761 caactacgaa aacaaatacg tagtgctgct aaaaagaata acttaccttt taagttgaca
8821 tgtgcaacta ctagacaagt tgttaatgtt gtaacaacaa agatagcact taagggtggt
8881 aaaattgtta ataattggtt gaagcagtta attaaagtta cacttgtgtt cctttttgtt
8941 gctgctattt tctatttaat aacacctgtt catgtcatgt ctaaacatac tgacttttca
9001 agtgaaatca taggatacaa ggctattgat ggtggtgtca ctcgtgacat agcatctaca
9061 gatacttgtt ttgctaacaa acatgctgat tttgacacat ggtttagcca gcgtggtggt
9121 agttatacta atgacaaagc ttgcccattg attgctgcag tcataacaag agaagtgggt
9181 tttgtcgtgc ctggtttgcc tggcacgata ttacgcacaa ctaatggtga ctttttgcat
9241 ttcttaccta gagtttttag tgcagttggt aacatctgtt acacaccatc aaaacttata
9301 gagtacactg actttgcaac atcagcttgt gttttggctg ctgaatgtac aatttttaaa
9361 gatgcttctg gtaagccagt accatattgt tatgatacca atgtactaga aggttctgtt
9421 gcttatgaaa gtttacgccc tgacacacgt tatgtgctca tggatggctc tattattcaa
9481 tttcctaaca cctaccttga aggttctgtt agagtggtaa caacttttga ttctgagtac
9541 tgtaggcacg gcacttgtga aagatcagaa gctggtgttt gtgtatctac tagtggtaga
9601 tgggtactta acaatgatta ttacagatct ttaccaggag ttttctgtgg tgtagatgct
9661 gtaaatttac ttactaatat gtttacacca ctaattcaac ctattggtgc tttggacata
9721 tcagcatcta tagtagctgg tggtattgta gctatcgtag taacatgcct tgcctactat
9781 tttatgaggt ttagaagagc ttttggtgaa tacagtcatg tagttgcctt taatacttta
9841 ctattcctta tgtcattcac tgtactctgt ttaacaccag tttactcatt cttacctggt
9901 gtttattctg ttatttactt gtacttgaca ttttatctta ctaatgatgt ttctttttta
9961 gcacatattc agtggatggt tatgttcaca cctttagtac ctttctggat aacaattgct
10021 tatatcattt gtatttccac aaagcatttc tattggttct ttagtaatta cctaaagaga
10081 cgtgtagtct ttaatggtgt ttcctttagt acttttgaag aagctgcgct gtgcaccttt
10141 ttgttaaata aagaaatgta tctaaagttg cgtagtgatg tgctattacc tcttacgcaa
10201 tataatagat acttagctct ttataataag tacaagtatt ttagtggagc aatggataca
10261 actagctaca gagaagctgc ttgttgtcat ctcgcaaagg ctctcaatga cttcagtaac
10321 tcaggttctg atgttcttta ccaaccacca caaacctcta tcacctcagc tgttttgcag
10381 agtggtttta gaaaaatggc attcccatct ggtaaagttg agggttgtat ggtacaagta
10441 acttgtggta caactacact taacggtctt tggcttgatg acgtagttta ctgtccaaga
10501 catgtgatct gcacctctga agacatgctt aaccctaatt atgaagattt actcattcgt
10561 aagtctaatc ataatttctt ggtacaggct ggtaatgttc aactcagggt tattggacat
10621 tctatgcaaa attgtgtact taagcttaag gttgatacag ccaatcctaa gacacctaag
10681 tataagtttg ttcgcattca accaggacag actttttcag tgttagcttg ttacaatggt
10741 tcaccatctg gtgtttacca atgtgctatg aggcccaatt tcactattaa gggttcattc
10801 cttaatggtt catgtggtag tgttggtttt aacatagatt atgactgtgt ctctttttgt
10861 tacatgcacc atatggaatt accaactgga gttcatgctg gcacagactt agaaggtaac
10921 ttttatggac cttttgttga caggcaaaca gcacaagcag ctggtacgga cacaactatt
10981 acagttaatg ttttagcttg gttgtacgct gctgttataa atggagacag gtggtttctc
11041 aatcgattta ccacaactct taatgacttt aaccttgtgg ctatgaagta caattatgaa
11101 cctctaacac aagaccatgt tgacatacta ggacctcttt ctgctcaaac tggaattgcc
11161 gttttagata tgtgtgcttc attaaaagaa ttactgcaaa atggtatgaa tggacgtacc
11221 atattgggta gtgctttatt agaagatgaa tttacacctt ttgatgttgt tagacaatgc 11281 tcaggtgtta ctttccaaag tgcagtgaaa agaacaatca agggtacaca ccactggttg
11341 ttactcacaa ttttgacttc acttttagtt ttagtccaga gtactcaatg gtctttgttc
11401 ttttttttgt atgaaaatgc ctttttacct tttgctatgg gtattattgc tatgtctgct
11461 tttgcaatga tgtttgtcaa acataagcat gcatttctct gtttgttttt gttaccttct
11521 cttgccactg tagcttattt taatatggtc tatatgcctg ctagttgggt gatgcgtatt
11581 atgacatggt tggatatggt tgatactagt ttgtctggtt ttaagctaaa agactgtgtt
11641 atgtatgcat cagctgtagt gttactaatc cttatgacag caagaactgt gtatgatgat
11701 ggtgctagga gagtgtggac acttatgaat gtcttgacac tcgtttataa agtttattat
11761 ggtaatgctt tagatcaagc catttccatg tgggctctta taatctctgt tacttctaac
11821 tactcaggtg tagttacaac tgtcatgttt ttggccagag gtattgtttt tatgtgtgtt
11881 gagtattgcc ctattttctt cataactggt aatacacttc agtgtataat gctagtttat
11941 tgtttcttag gctatttttg tacttgttac tttggcctct tttgtttact caaccgctac
12001 tttagactga ctcttggtgt ttatgattac ttagtttcta cacaggagtt tagatatatg
12061 aattcacagg gactactccc acccaagaat agcatagatg ccttcaaact caacattaaa
12121 ttgttgggtg ttggtggcaa accttgtatc aaagtagcca ctgtacagtc taaaatgtca
12181 gatgtaaagt gcacatcagt agtcttactc tcagttttgc aacaactcag agtagaatca
12241 tcatctaaat tgtgggctca atgtgtccag ttacacaatg acattctctt agctaaagat
12301 actactgaag cctttgaaaa aatggtttca ctactttctg ttttgctttc catgcagggt
12361 gctgtagaca taaacaagct ttgtgaagaa atgctggaca acagggcaac cttacaagct
12421 atagcctcag agtttagttc ccttccatca tatgcagctt ttgctactgc tcaagaagct
12481 tatgagcagg ctgttgctaa tggtgattct gaagttgttc ttaaaaagtt gaagaagtct
12541 ttgaatgtgg ctaaatctga atttgaccgt gatgcagcca tgcaacgtaa gttggaaaag
12601 atggctgatc aagctatgac ccaaatgtat aaacaggcta gatctgagga caagagggca
12661 aaagttacta gtgctatgca gacaatgctt ttcactatgc ttagaaagtt ggataatgat
12721 gcactcaaca acattatcaa caatgcaaga gatggttgtg ttcccttgaa cataatacct
12781 cttacaacag cagccaaact aatggttgtc ataccagact ataacacata taaaaatacg
12841 tgtgatggta caacatttac ttatgcatca gcattgtggg aaatccaaca ggttgtagat
12901 gcagatagta aaattgttca acttagtgaa attagtatgg acaattcacc taatttagca
12961 tggcctctta ttgtaacagc tttaagggcc aattctgctg tcaaattaca gaataatgag
13021 cttagtcctg ttgcactacg acagatgtct tgtgctgccg gtactacaca aactgcttgc
13081 actgatgaca atgcgttagc ttactacaac acaacaaagg gaggtaggtt tgtacttgca
13141 ctgttatccg atttacagga tttgaaatgg gctagattcc ctaagagtga tggaactggt
13201 actatctata cagaactgga accaccttgt aggtttgtta cagacacacc taaaggtcct
13261 aaagtgaagt atttatactt tattaaagga ttaaacaacc taaatagagg tatggtactt
13321 ggtagtttag ctgccacagt acgtctacaa gctggtaatg caacagaagt gcctgccaat
13381 tcaactgtat tatctttctg tgcttttgct gtagatgctg ctaaagctta caaagattat
13441 ctagctagtg ggggacaacc aatcactaat tgtgttaaga tgttgtgtac acacactggt
13501 actggtcagg caataacagt tacaccggaa gccaatatgg atcaagaatc ctttggtggt
13561 gcatcgtgtt gtctgtactg ccgttgccac atagatcatc caaatcctaa aggattttgt
13621 gacttaaaag gtaagtatgt acaaatacct acaacttgtg ctaatgaccc tgtgggtttt
13681 acacttaaaa acacagtctg taccgtctgc ggtatgtgga aaggttatgg ctgtagttgt
13741 gatcaactcc gcgaacccat gcttcagtca gctgatgcac aatcgttttt aaacgggttt
13801 gcggtgtaag tgcagcccgt cttacaccgt gcggcacagg cactagtact gatgtcgtat
13861 acagggcttt tgacatctac aatgataaag tagctggttt tgctaaattc ctaaaaacta
13921 attgttgtcg cttccaagaa aaggacgaag atgacaattt aattgattct tactttgtag
13981 ttaagagaca cactttctct aactaccaac atgaagaaac aatttataat ttacttaagg
14041 attgtccagc tgttgctaaa catgacttct ttaagtttag aatagacggt gacatggtac
14101 cacatatatc acgtcaacgt cttactaaat acacaatggc agacctcgtc tatgctttaa
14161 ggcattttga tgaaggtaat tgtgacacat taaaagaaat acttgtcaca tacaattgtt
14221 gtgatgatga ttatttcaat aaaaaggact ggtatgattt tgtagaaaac ccagatatat
14281 tacgcgtata cgccaactta ggtgaacgtg tacgccaagc tttgttaaaa acagtacaat
14341 tctgtgatgc catgcgaaat gctggtattg ttggtgtact gacattagat aatcaagatc
14401 tcaatggtaa ctggtatgat ttcggtgatt tcatacaaac cacgccaggt agtggagttc
14461 ctgttgtaga ttcttattat tcattgttaa tgcctatatt aaccttgacc agggctttaa
14521 ctgcagagtc acatgttgac actgacttaa caaagcctta cattaagtgg gatttgttaa
14581 aatatgactt cacggaagag aggttaaaac tctttgaccg ttattttaaa tattgggatc
14641 agacatacca cccaaattgt gttaactgtt tggatgacag atgcattctg cattgtgcaa 14701 actttaatgt tttattctct acagtgttcc cacctacaag ttttggacca ctagtgagaa
14761 aaatatttgt tgatggtgtt ccatttgtag tttcaactgg ataccacttc agagagctag
14821 gtgttgtaca taatcaggat gtaaacttac atagctctag acttagtttt aaggaattac
14881 ttgtgtatgc tgctgaccct gctatgcacg ctgcttctgg taatctatta ctagataaac
14941 gcactacgtg cttttcagta gctgcactta ctaacaatgt tgcttttcaa actgtcaaac
15001 ccggtaattt taacaaagac ttctatgact ttgctgtgtc taagggtttc tttaaggaag
15061 gaagttctgt tgaattaaaa cacttcttct ttgctcagga tggtaatgct gctatcagcg
15121 attatgacta ctatcgttat aatctaccaa caatgtgtga tatcagacaa ctactatttg
15181 tagttgaagt tgttgataag tactttgatt gttacgatgg tggctgtatt aatgctaacc
15241 aagtcatcgt caacaaccta gacaaatcag ctggttttcc atttaataaa tggggtaagg
15301 ctagacttta ttatgattca atgagttatg aggatcaaga tgcacttttc gcatatacaa
15361 aacgtaatgt catccctact ataactcaaa tgaatcttaa gtatgccatt agtgcaaaga
15421 atagagctcg caccgtagct ggtgtctcta tctgtagtac tatgaccaat agacagtttc
15481 atcaaaaatt attgaaatca atagccgcca ctagaggagc tactgtagta attggaacaa
15541 gcaaattcta tggtggttgg cacaacatgt taaaaactgt ttatagtgat gtagaaaacc
15601 ctcaccttat gggttgggat tatcctaaat gtgatagagc catgcctaac atgcttagaa
15661 ttatggcctc acttgttctt gctcgcaaac atacaacgtg ttgtagcttg tcacaccgtt
15721 tctatagatt agctaatgag tgtgctcaag tattgagtga aatggtcatg tgtggcggtt
15781 cactatatgt taaaccaggt ggaacctcat caggagatgc cacaactgct tatgctaata
15841 gtgtttttaa catttgtcaa gctgtcacgg ccaatgttaa tgcactttta tctactgatg
15901 gtaacaaaat tgccgataag tatgtccgca atttacaaca cagactttat gagtgtctct
15961 atagaaatag agatgttgac acagactttg tgaatgagtt ttacgcatat ttgcgtaaac
16021 atttctcaat gatgatactc tctgacgatg ctgttgtgtg tttcaatagc acttatgcat
16081 ctcaaggtct agtggctagc ataaagaact ttaagtcagt tctttattat caaaacaatg
16141 tttttatgtc tgaagcaaaa tgttggactg agactgacct tactaaagga cctcatgaat
16201 tttgctctca acatacaatg ctagttaaac agggtgatga ttatgtgtac cttccttacc
16261 cagatccatc aagaatccta ggggccggct gttttgtaga tgatatcgta aaaacagatg
16321 gtacacttat gattgaacgg ttcgtgtctt tagctataga tgcttaccca cttactaaac
16381 atcctaatca ggagtatgct gatgtctttc atttgtactt acaatacata agaaagctac
16441 atgatgagtt aacaggacac atgttagaca tgtattctgt tatgcttact aatgataaca
16501 cttcaaggta ttgggaacct gagttttatg aggctatgta cacaccgcat acagtcttac
16561 aggctgttgg ggcttgtgtt ctttgcaatt cacagacttc attaagatgt ggtgcttgca
16621 tacgtagacc attcttatgt tgtaaatgct gttacgacca tgtcatatca acatcacata
16681 aattagtctt gtctgttaat ccgtatgttt gcaatgctcc aggttgtgat gtcacagatg
16741 tgactcaact ttacttagga ggtatgagct attattgtaa atcacataaa ccacccatta
16801 gttttccatt gtgtgctaat ggacaagttt ttggtttata taaaaataca tgtgttggta
16861 gcgataatgt tactgacttt aatgcaattg caacatgtga ctggacaaat gctggtgatt
16921 acattttagc taacacctgt actgaaagac tcaagctttt tgcagcagaa acgctcaaag
16981 ctactgagga gacatttaaa ctgtcttatg gtattgctac tgtacgtgaa gtgctgtctg
17041 acagagaatt acatctttca tgggaagttg gtaaacctag accaccactt aaccgaaatt
17101 atgtctttac tggttatcgt gtaactaaaa acagtaaagt acaaatagga gagtacacct
17161 ttgaaaaagg tgactatggt gatgctgttg tttaccgagg tacaacaact tacaaattaa
17221 atgttggtga ttattttgtg ctgacatcac atacagtaat gccattaagt gcacctacac
17281 tagtgccaca agagcactat gttagaatta ctggcttata cccaacactc aatatctcag
17341 atgagttttc tagcaatgtt gcaaattatc aaaaggttgg tatgcaaaag tattctacac
17401 tccagggacc acctggtact ggtaagagtc attttgctat tggcctagct ctctactacc
17461 cttctgctcg catagtgtat acagcttgct ctcatgccgc tgttgatgca ctatgtgaga
17521 aggcattaaa atatttgcct atagataaat gtagtagaat tatacctgca cgtgctcgtg
17581 tagagtgttt tgataaattc aaagtgaatt caacattaga acagtatgtc ttttgtactg
17641 taaatgcatt gcctgagacg acagcagata tagttgtctt tgatgaaatt tcaatggcca
17701 caaattatga tttgagtgtt gtcaatgcca gattacgtgc taagcactat gtgtacattg
17761 gcgaccctgc tcaattacct gcaccacgca cattgctaac taagggcaca ctagaaccag
17821 aatatttcaa ttcagtgtgt agacttatga aaactatagg tccagacatg ttcctcggaa
17881 cttgtcggcg ttgtcctgct gaaattgttg acactgtgag tgctttggtt tatgataata
17941 agcttaaagc acataaagac aaatcagctc aatgctttaa aatgttttat aagggtgtta
18001 tcacgcatga tgtttcatct gcaattaaca ggccacaaat aggcgtggta agagaattcc
18061 ttacacgtaa ccctgcttgg agaaaagctg tctttatttc accttataat tcacagaatg 18121 ctgtagcctc aaagattttg ggactaccaa ctcaaactgt tgattcatca cagggctcag
18181 aatatgacta tgtcatattc actcaaacca ctgaaacagc tcactcttgt aatgtaaaca
18241 gatttaatgt tgctattacc agagcaaaag taggcatact ttgcataatg tctgatagag
18301 acctttatga caagttgcaa tttacaagtc ttgaaattcc acgtaggaat gtggcaactt
18361 tacaagctga aaatgtaaca ggactcttta aagattgtag taaggtaatc actgggttac
18421 atcctacaca ggcacctaca cacctcagtg ttgacactaa attcaaaact gaaggtttat
18481 gtgttgacat acctggcata cctaaggaca tgacctatag aagactcatc tctatgatgg
18541 gttttaaaat gaattatcaa gttaatggtt accctaacat gtttatcacc cgcgaagaag
18601 ctataagaca tgtacgtgca tggattggct tcgatgtcga ggggtgtcat gctactagag
18661 aagctgttgg taccaattta cctttacagc taggtttttc tacaggtgtt aacctagttg
18721 ctgtacctac aggttatgtt gatacaccta ataatacaga tttttccaga gttagtgcta
18781 aaccaccgcc tggagatcaa tttaaacacc tcataccact tatgtacaaa ggacttcctt
18841 ggaatgtagt gcgtataaag attgtacaaa tgttaagtga cacacttaaa aatctctctg
18901 acagagtcgt atttgtctta tgggcacatg gctttgagtt gacatctatg aagtattttg
18961 tgaaaatagg acctgagcgc acctgttgtc tatgtgatag acgtgccaca tgcttttcca
19021 ctgcttcaga cacttatgcc tgttggcatc attctattgg atttgattac gtctataatc
19081 cgtttatgat tgatgttcaa caatggggtt ttacaggtaa cctacaaagc aaccatgatc
19141 tgtattgtca agtccatggt aatgcacatg tagctagttg tgatgcaatc atgactaggt
19201 gtctagctgt ccacgagtgc tttgttaagc gtgttgactg gactattgaa tatcctataa
19261 ttggtgatga actgaagatt aatgcggctt gtagaaaggt tcaacacatg gttgttaaag
19321 ctgcattatt agcagacaaa ttcccagttc ttcacgacat tggtaaccct aaagctatta
19381 agtgtgtacc tcaagctgat gtagaatgga agttctatga tgcacagcct tgtagtgaca
19441 aagcttataa aatagaagaa ttattctatt cttatgccac acattctgac aaattcacag
19501 atggtgtatg cctattttgg aattgcaatg tcgatagata tcctgctaat tccattgttt
19561 gtagatttga cactagagtg ctatctaacc ttaacttgcc tggttgtgat ggtggcagtt
19621 tgtatgtaaa taaacatgca ttccacacac cagcttttga taaaagtgct tttgttaatt
19681 taaaacaatt accatttttc tattactctg acagtccatg tgagtctcat ggaaaacaag
19741 tagtgtcaga tatagattat gtaccactaa agtctgctac gtgtataaca cgttgcaatt
19801 taggtggtgc tgtctgtaga catcatgcta atgagtacag attgtatctc gatgcttata
19861 acatgatgat ctcagctggc tttagcttgt gggtttacaa acaatttgat acttataacc
19921 tctggaacac ttttacaaga cttcagagtt tagaaaatgt ggcttttaat gttgtaaata
19981 agggacactt tgatggacaa cagggtgaag taccagtttc tatcattaat aacactgttt
20041 acacaaaagt tgatggtgtt gatgtagaat tgtttgaaaa taaaacaaca ttacctgtta
20101 atgtagcatt tgagctttgg gctaagcgca acattaaacc agtaccagag gtgaaaatac
20161 tcaataattt gggtgtggac attgctgcta atactgtgat ctgggactac aaaagagatg
20221 ctccagcaca tatatctact attggtgttt gttctatgac tgacatagcc aagaaaccaa
20281 ctgaaacgat ttgtgcacca ctcactgtct tttttgatgg tagagttgat ggtcaagtag
20341 acttatttag aaatgcccgt aatggtgttc ttattacaga aggtagtgtt aaaggtttac
20401 aaccatctgt aggtcccaaa caagctagtc ttaatggagt cacattaatt ggagaagccg
20461 taaaaacaca gttcaattat tataagaaag ttgatggtgt tgtccaacaa ttacctgaaa
20521 cttactttac tcagagtaga aatttacaag aatttaaacc caggagtcaa atggaaattg
20581 atttcttaga attagctatg gatgaattca ttgaacggta taaattagaa ggctatgcct
20641 tcgaacatat cgtttatgga gattttagtc atagtcagtt aggtggttta catctactga
20701 ttggactagc taaacgtttt aaggaatcac cttttgaatt agaagatttt attcctatgg
20761 acagtacagt taaaaactat ttcataacag atgcgcaaac aggttcatct aagtgtgtgt
20821 gttctgttat tgatttatta cttgatgatt ttgttgaaat aataaaatcc caagatttat
20881 ctgtagtttc taaggttgtc aaagtgacta ttgactatac agaaatttca tttatgcttt
20941 ggtgtaaaga tggccatgta gaaacatttt acccaaaatt acaatctagt caagcgtggc
21001 aaccgggtgt tgctatgcct aatctttaca aaatgcaaag aatgctatta gaaaagtgtg
21061 accttcaaaa ttatggtgat agtgcaacat tacctaaagg cataatgatg aatgtcgcaa
21121 aatatactca actgtgtcaa tatttaaaca cattaacatt agctgtaccc tataatatga
21181 gagttataca ttttggtgct ggttctgata aaggagttgc accaggtaca gctgttttaa
21241 gacagtggtt gcctacgggt acgctgcttg tcgattcaga tcttaatgac tttgtctctg
21301 atgcagattc aactttgatt ggtgattgtg caactgtaca tacagctaat aaatgggatc
21361 tcattattag tgatatgtac gaccctaaga ctaaaaatgt tacaaaagaa aatgactcta
21421 aagagggttt tttcacttac atttgtgggt ttatacaaca aaagctagct cttggaggtt
21481 ccgtggctat aaagataaca gaacattctt ggaatgctga tctttataag ctcatgggac 21541 acttcgcatg gtggacagcc tttgttacta atgtgaatgc gtcatcatct gaagcatttt 21601 taattggatg taattatctt ggcaaaccac gcgaacaaat agatggttat gtcatgcatg 21661 caaattacat attttggagg aatacaaatc caattcagtt gtcttcctat tctttatttg 21721 acatgagtaa atttcccctt aaattaaggg gtactgctgt tatgtcttta aaagaaggtc 21781 aaatcaatga tatgatttta tctcttctta gtaaaggtag acttataatt agagaaaaca 21841 acagagttgt tatttctagt gatgttcttg ttaacaacta aacgaacaAT GGTTATTGAA 21901 CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC 21961 TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG 22021 CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC TGAATGAACT GCAGGACGAG 22081 GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT 22141 GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG 22201 TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG 22261 CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA 22321 GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG 22381 GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC GCATGCCCGA CGGCGAGGAT 22441 CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT 22501 TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG 22561 GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT 22621 TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC 22681 TTCGAGGGCA GAGGAAGTCT TCTAACATGC GGTGACGTGG AGGAGAATCC CGGCCCTATG 22741 GAcCCGAAGA AAAAgCGTAA AGTGGACCCt AAGAAgAAAC GcAAAGTTGA TCCGAAGAAA 22801 AAACGTAAAG TGGTGAGCAA GGGCGAGGAG GATAACATGG CCTCTCTCCC AGCGACACAT 22861 GAGTTACACA TCTTTGGCTC CATCAACGGT GTGGACTTTG ACATGGTGGG TCAGGGCACC 22921 GGCAATCCAA ATGATGGTTA TGAGGAGTTA AACCTGAAGT CCACCAAGGG TGACCTCCAG 22981 TTCTCCCCCT GGATTCTGGT CCCTCATATC GGGTATGGCT TCCATCAGTA CCTGCCCTAC 23041 CCTGACGGGA TGTCGCCTTT CCAGGCCGCC ATGGTAGATG GCTCCGGATA CCAAGTCCAT 23101 CGCACAATGC AGTTTGAAGA TGGTGCCTCC CTTACTGTTA ACTACCGCTA CACCTACGAG 23161 GGAAGCCACA TCAAAGGAGA GGCCCAGGTG AAGGGGACTG GTTTCCCTGC TGACGGTCCT 23221 GTGATGACCA ACTCGCTGAC CGCTGCGGAC TGGTGCAGGT CGAAGAAGAC TTACCCCAAC 23281 GACAAAACCA TCATCAGTAC CTTTAAGTGG AGTTACACCA CTGGAAATGG CAAGCGCTAC 23341 CGGAGCACTG CGCGGACCAC CTACACCTTT GCCAAGCCAA TGGCGGCTAA CTATCTGAAG 23401 AACCAGCCGA TGTACGTGTT CCGTAAGACG GAGCTCAAGC ACTCCAAGAC CGAGCTCAAC 23461 TTCAAGGAGT GGCAAAAGGC CTTTACCGAT GTGATGGGCA TGGACGAGCT GTACAAGTAA 23521 acgaacttat ggatttgttt atgagaatct tcacaattgg aactgtaact ttgaagcaag 23581 gtgaaatcaa ggatgctact ccttcagatt ttgttcgcgc tactgcaacg ataccgatac 23641 aagcctcact ccctttcgga tggcttattg ttggcgttgc acttcttgct gtttttcaga 23701 gcgcttccaa aatcataacc ctcaaaaaga gatggcaact agcactctcc aagggtgttc 23761 actttgtttg caacttgctg ttgttgtttg taacagttta ctcacacctt ttgctcgttg 23821 ctgctggcct tgaagcccct tttctctatc tttatgcttt agtctacttc ttgcagagta 23881 taaactttgt aagaataata atgaggcttt ggctttgctg gaaatgccgt tccaaaaacc 23941 cattacttta tgatgccaac tattttcttt gctggcatac taattgttac gactattgta 24001 taccttacaa tagtgtaact tcttcaattg tcattacttc aggtgatggc acaacaagtc 24061 ctatttctga acatgactac cagattggtg gttatactga aaaatgggaa tctggagtaa 24121 aagactgtgt tgtattacac agttacttca cttcagacta ttaccagctg tactcaactc 24181 aattgagtac agacactggt gttgaacatg ttaccttctt catctacaat aaaattgttg 24241 atgagcctga agaacatgtc caaattcaca caatcgacgg ttcatccgga gttgttaatc 24301 cagtaatgga accaatttat gatgaaccga cgacgactac tagcgtgcct ttgtaagcac 24361 aagctgatga gtacgaactt atgtactcat tcgtttcgga agagacaggt acgttaatag 24421 ttaatagcgt acttcttttt cttgctttcg tggtattctt gctagttaca ctagccatcc 24481 ttactgcgct tcgattgtgt gcgtactgct gcaatattgt taacgtgagt cttgtaaaac 24541 cttcttttta cgtttactct cgtgttaaaa atctgaattc ttctagagtt cctgatcttc 24601 tggtctaaac gaactaaata ttatattagt ttttctgttt ggaactttaa ttttagccat 24661 ggcagattcc aacggtacta ttaccgttga agagcttaaa aagctccttg aacaatggaa 24721 cctagtaata ggtttcctat tccttacatg gatttgtctt ctacaatttg cctatgccaa 24781 caggaatagg tttttgtata taattaagtt aattttcctc tggctgttat ggccagtaac 24841 tttagcttgt tttgtgcttg ctgctgttta cagaataaat tggatcaccg gtggaattgc 24901 tatcgcaatg gcttgtcttg taggcttgat gtggctcagc tacttcattg cttctttcag 24961 actgtttgcg cgtacgcgtt ccatgtggtc attcaatcca gaaactaaca ttcttctcaa
25021 cgtgccactc catggcacta ttctgaccag accgcttcta gaaagtgaac tcgtaatcgg
25081 agctgtgatc cttcgtggac atcttcgtat tgctggacac catctaggac gctgtgacat
25141 caaggacctg cctaaagaaa tcactgttgc tacatcacga acgctttctt attacaaatt
25201 gggagcttcg cagcgtgtag caggtgactc aggttttgct gcatacagtc gctacaggat
25261 tggcaactat aaattaaaca cagaccattc cagtagcagt gacaatattg ctttgcttgt
25321 acagtaagtg acaacagatg tttcatctcg ttgactttca ggttactata gcagagatat
25381 tactaattat tatgaggact tttaaagttt ccatttggaa tcttgattac atcataaacc
25441 tcataattaa aaatttatct aagtcactaa ctgagaataa atattctcaa ttagatgaag
25501 agcaaccaat ggagattgat taaacgaaca tgaaaattat tcttttcttg gcactgataa
25561 cactcgctac ttgtgagctt tatcactacc aagagtgtgt tagaggtaca acagtacttt
25621 taaaagaacc ttgctcttct ggaacatacg agggcaattc accatttcat cctctagctg
25681 ataacaaatt tgcactgact tgctttagca ctcaatttgc ttttgcttgt cctgacggcg
25741 taaaacacgt ctatcagtta cgtgccagat cagtttcacc taaactgttc atcagacaag
25801 aggaagttca agaactttac tctccaattt ttcttattgt tgcggcaata gtgtttataa
25861 cactttgctt cacactcaaa agaaagacag aatgattgaa ctttcattaa ttgacttcta
25921 tttgtgcttt ttagcctttc tgctattcct tgttttaatt atgcttatta tcttttggtt
25981 ctcacttgaa ctgcaagatc ataatgaaac ttgtcacgcc taaacgaaca tgaaatttct
26041 tgttttctta ggaatcatca caactgtagc tgcatttcac caagaatgta gtttacagtc
26101 atgtactcaa catcaaccat atgtagttga tgacccgtgt cctattcact tctattctaa
26161 atggtatatt agagtaggag ctagaaaatc agcaccttta attgaattgt gcgtggatga
26221 ggctggttct aaatcaccca ttcagtacat cgatatcggt aattatacag tttcctgttt
26281 accttttaca attaattgcc aggaacctaa attgggtagt cttgtagtgc gttgttcgtt
26341 ctatgaagac tttttagagt atcatgacgt tcgtgttgtt ttagatttca tctaaacgaa
26401 caaactaaaa tgtctgataa tggaccccaa aatcagcgaa atgcaccccg cattacgttt
26461 ggtggaccct cagattcaac tggcagtaac cagaatggag aacgcagtgg ggcgcgatca
26521 aaacaacgtc ggccccaagg tttacccaat aatactgcgt cttggttcac cgctctcact
26581 caacatggca aggaagacct taaattccct cgaggacaag gcgttccaat taacaccaat
26641 agcagtccag atgaccaaat tggctactac cgaagagcta ccagacgaat tcgtggtggt
26701 gacggtaaaa tgaaagatct cagtccaaga tggtatttct actacctagg aactgggcca
26761 gaagctggac ttccctatgg tgctaacaaa gacggcatca tatgggttgc aactgaggga
26821 gccttgaata caccaaaaga tcacattggc acccgcaatc ctgctaacaa tgctgcaatc
26881 gtgctacaac ttcctcaagg aacaacattg ccaaaaggct tctacgcaga agggagcaga
26941 ggcggcagtc aagcctcttc tcgttcctca tcacgtagtc gcaacagttc aagaaattca
27001 actccaggca gcagtagggg aacttctcct gctagaatgg ctggcaatgg cggtgatgct
27061 gctcttgctt tgctgctgct tgacagattg aaccagcttg agagcaaaat gtctggtaaa
27121 ggccaacaac aacaaggcca aactgtcact aagaaatctg ctgctgaggc ttctaagaag
27181 cctcggcaaa aacgtactgc cactaaagca tacaatgtaa cacaagcttt cggcagacgt
27241 ggtccagaac aaacccaagg aaattttggg gaccaggaac taatcagaca aggaactgat
27301 tacaaacatt ggccgcaaat tgcacaattt gcccccagcg cttcagcgtt cttcggaatg
27361 tcgcgcattg gcatggaagt cacaccttcg ggaacgtggt tgacctacac aggtgccatc
27421 aaattggatg acaaagatcc aaatttcaaa gatcaagtca ttttgctgaa taagcatatt
27481 gacgcataca aaacattccc accaacagag cctaaaaagg acaaaaagaa gaaggctgat
27541 gaaactcaag ccttaccgca gagacagaag aaacagcaaa ctgtgactct tcttcctgct
27601 gcagatttgg atgatttctc caaacaattg caacaatcca tgagcagtgc tgactcaact
27661 caggcctaaa ctcatgcaga ccacacaagg cagatgggct atataaacgt tttcgctttt
27721 ccgtttacga tatatagtct actcttgtgc agaatgaatt ctcgtaacta catagcacaa
27781 gtagatgtag ttaactttaa tctcacatag caatctttaa tcagtgtgta acattaggga
27841 ggacttgaaa gagccaccac attttcaccg aggccacgcg gagtacgatc gagtgtacag
27901 tgaacaatgc tagggagagc tgcctatatg gaagagccct aatgtgtaaa attaatttta
27961 gtagtgctat ccccatgtga ttttaatagc ttcttaggag aatgacaaaa aaaaaaaaaa
28021 aaaaaaggcc ggcatggtcc cagcctcctc gctggcgccg gctgggcaac attccgaggg
28081 gaccgtcccc tcggtaatgg cgaatgggac GGGCCCTGCG ATATCGCGAC GAGGATCTAg
28141 atcctctaga gtcgacctgc aggcatgcaa gcttgagtat tctatagtct cacctaaata
28201 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
28261 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
28321 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 28381 agctgcatta atgaatcggc caacgcgaac cccttgcggc cgcccgggcc gtcgaccaat 28441 tctcatgttt gacagcttat catcgaattt ctgccattca tccgcttatt atcacttatt 28501 caggcgtagc aaccaggcgt ttaagggcac caataactgc cttaaaaaaa ttacgccccg 28561 ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca 28621 tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta 28681 taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc cacgtttaaa 28741 tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt ctcaataaac 28801 cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga atatatgtgt 28861 agaaactgcc ggaaatcgtc gtggtattca ctccagagcg atgaaaacgt ttcagtttgc 28921 tcatggaaaa eggtgtaaca agggtgaaca ctatcccata tcaccagctc accgtctttc 28981 attgccatac gaaattccgg atgagcattc atcaggcggg caagaatgtg aataaaggcc 29041 ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat atccagctga 29101 acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga 29161 tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc 29221 ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta 29281 tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca aaagttggcc 29341 cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa gtgatcttcc 29401 gtcacaggta tttattcgcg ataagctcat ggagcggcgt aaccgtcgca caggaaggac 29461 agagaaagcg cggatctggg aagtgacgga cagaacggtc aggacctgga ttggggaggc 29521 ggttgccgcc gctgctgctg acggtgtgac gttctctgtt ccggtcacac cacatacgtt 29581 ccgccattcc tatgcgatgc acatgctgta tgccggtata ccgctgaaag ttctgcaaag 29641 cctgatggga cataagtcca tcagttcaac ggaagtctac acgaaggttt ttgcgctgga 29701 tgtggctgcc cggcaccggg tgcagtttgc gatgccggag tctgatgcgg ttgcgatgct 29761 gaaacaatta tcctgagaat aaatgccttg gcctttatat ggaaatgtgg aactgagtgg 29821 atatgctgtt tttgtctgtt aaacagagaa gctggctgtt atccactgag aagcgaacga 29881 aacagtcggg aaaatctccc attatcgtag agatccgcat tattaatctc aggagcctgt 29941 gtagcgttta taggaagtag tgttctgtca tgatgcctgc aagcggtaac gaaaacgatt 30001 tgaatatgcc ttcaggaaca atagaaatct tcgtgcggtg ttacgttgaa gtggagcgga 30061 ttatgtcagc aatggacaga acaacctaat gaacacagaa ccatgatgtg gtctgtcctt 30121 ttacagccag tagtgctcgc cgcagtcgag cgacagggcg aagccctcga gctggttgcc 30181 ctcgccgctg ggctggcggc cgtctatggc cctgcaaacg cgccagaaac gccgtcgaag 30241 ccgtgtgcga gacaccgcgg ccggccgccg gcgttgtgga tacctcgcgg aaaacttggc 30301 cctcactgac agatgagggg cggacgttga cacttgaggg gccgactcac ccggcgcggc 30361 gttgacagat gaggggcagg ctcgatttcg gccggcgacg tggagctggc cagcctcgca 30421 aatcggcgaa aacgcctgat tttacgcgag tttcccacag atgatgtgga caagcctggg 30481 gataagtgcc ctgcggtatt gacacttgag gggcgcgact actgacagat gaggggcgcg 30541 atccttgaca cttgaggggc agagtgctga cagatgaggg gcgcacctat tgacatttga 30601 ggggctgtcc acaggcagaa aatccagcat ttgcaagggt ttccgcccgt ttttcggcca 30661 ccgctaacct gtcttttaac ctgcttttaa accaatattt ataaaccttg tttttaacca 30721 gggctgcgcc ctgtgcgcgt gaccgcgcac gccgaagggg ggtgcccccc cttctcgaac 30781 cctcccggtc gagtgagcga ggaagcacca gggaacagca cttatatatt ctgcttacac 30841 acgatgcctg aaaaaacttc ccttggggtt atccacttat ccacggggat atttttataa 30901 ttattttttt tatagttttt agatcttctt ttttagagcg ccttgtaggc ctttatccat 30961 gctggttcta gagaaggtgt tgtgacaaat tgccctttca gtgtgacaaa tcaccctcaa 31021 atgacagtcc tgtctgtgac aaattgccct taaccctgtg acaaattgcc ctcagaagaa 31081 gctgtttttt cacaaagtta tccctgctta ttgactcttt tttatttagt gtgacaatct 31141 aaaaacttgt cacacttcac atggatctgt catggcggaa acagcggtta tcaatcacaa 31201 gaaacgtaaa aatagcccgc gaatcgtcca gtcaaacgac ctcactgagg cggcatatag 31261 tctctcccgg gatcaaaaac gtatgctgta tctgttcgtt gaccagatca gaaaatctga 31321 tggcacccta caggaacatg acggtatctg cgagatccat gttgctaaat atgctgaaat 31381 attcggattg acctctgcgg aagccagtaa ggatatacgg caggcattga agagtttcgc 31441 ggggaaggaa gtggtttttt atcgccctga agaggatgcc ggcgatgaaa aaggctatga 31501 atcttttcct tggtttatca aacgtgcgca cagtccatcc agagggcttt acagtgtaca 31561 tatcaaccca tatctcattc ccttctttat cgggttacag aaccggttta cgcagtttcg 31621 gcttagtgaa acaaaagaaa tcaccaatcc gtatgccatg cgtttatacg aatccctgtg 31681 tcagtatcgt aagccggatg gctcaggcat cgtctctctg aaaatcgact ggatcataga 31741 gcgttaccag ctgcctcaaa gttaccagcg tatgcctgac ttccgccgcc gcttcctgca 31801 ggtctgtgtt aatgagatca acagcagaac tccaatgcgc ctctcataca ttgagaaaaa
31861 gaaaggccgc cagacgactc atatcgtatt ttccttccgc gatatcactt ccatgacgac
31921 aggatagtct gagggttatc tgtcacagat ttgagggtgg ttcgtcacat ttgttctgac
31981 ctactgaggg taatttgtca cagttttgct gtttccttca gcctgcatgg attttctcat
32041 actttttgaa ctgtaatttt taaggaagcc aaatttgagg gcagtttgtc acagttgatt
32101 tccttctctt tcccttcgtc atgtgacctg atatcggggg ttagttcgtc atcattgatg
32161 agggttgatt atcacagttt attactctga attggctatc cgcgtgtgta cctctacctg
32221 gagtttttcc cacggtggat atttcttctt gcgctgagcg taagagctat ctgacagaac
32281 agttcttctt tgcttcctcg ccagttcgct cgctatgctc ggttacacgg ctgcggcgag
32341 cgctagtgat aataagtgac tgaggtatgt gctcttctta tctccttttg tagtgttgct
32401 cttattttaa acaactttgc ggttttttga tgactttgcg attttgttgt tgctttgcag
32461 taaattgcaa gatttaataa aaaaacgcaa agcaatgatt aaaggatgtt cagaatgaaa
32521 ctcatggaaa cacttaacca gtgcataaac gctggtcatg aaatgacgaa ggctatcgcc
32581 attgcacagt ttaatgatga cagcccggaa gcgaggaaaa taacccggcg ctggagaata
32641 ggtgaagcag cggatttagt tggggtttct tctcaggcta tcagagatgc cgagaaagca
32701 gggcgactac cgcacccgga tatggaaatt cgaggacggg ttgagcaacg tgttggttat
32761 acaattgaac aaattaatca tatgcgtgat gtgtttggta cgcgattgcg acgtgctgaa
32821 gacgtatttc caccggtgat cqqqqttqct gcccataaag gtggcgttta caaaacctca
32881 gtttctgttc atcttgctca ggatctggct ctgaaggggc tacgtgtttt gctcgtggaa
32941 ggtaacgacc cccagggaac agcctcaatg tatcacggat gggtaccaga tcttcatatt
33001 catgcagaag acactctcct gcctttctat cttggggaaa aggacgatgt cacttatgca
33061 ataaagccca cttgctggcc ggggcttgac attattcctt cctgtctggc tctgcaccgt
33121 attgaaactg agttaatggg caaatttgat gaaggtaaac tgcccaccga tccacacctg
33181 atgctccgac tggccattga aactgttgct catgactatg atgtcatagt tattgacagc
33241 gcgcctaacc tgggtatcgg cacgattaat gtcgtatgtg ctgctgatgt gctgattgtt
33301 cccacgcctg ctgagttgtt tgactacacc tccgcactgc agtttttcga tatgcttcgt
33361 gatctgctca agaacgttga tcttaaaggg ttcgagcctg atgtacgtat tttgcttacc
33421 aaatacagca atagcaatgg ctctcagtcc ccgtggatgg aggagcaaat tcgggatgcc
33481 tggggaagca tggttctaaa aaatgttgta cgtgaaacgg atgaagttgg taaaggtcag
33541 atccggatga gaactgtttt tgaacaggcc attgatcaac gctcttcaac tggtgcctgg
33601 agaaatgctc tttctatttg ggaacctgtc tgcaatgaaa ttttcgatcg tctgattaaa
33661 ccacgctggg agattagata atgaagcgtg cgcctgttat tccaaaacat acgctcaata
33721 ctcaaccggt tgaagatact tcgttatcga caccagctgc cccgatggtg gattcgttaa
33781 ttgcgcgcgt aggagtaatg gctcgcggta atgccattac tttgcctgta tgtggtcggg
33841 atgtgaagtt tactcttgaa gtgctccggg gtgatagtgt tgagaagacc tctcgggtat
33901 ggtcaggtaa tgaacgtgac caggagctgc ttactgagga cgcactggat gatctcatcc
33961 cttcttttct actgactggt caacagacac cggcgttcgg tcgaagagta tctggtgtca
34021 tagaaattgc cgatgggagt cgccgtcgta aagctgctgc acttaccgaa agtgattatc
34081 gtgttctggt tggcgagctg gatgatgagc agatggctgc attatccaga ttgggtaacg
34141 attatcgccc aacaagtgct tatgaacgtg gtcagcgtta tgcaagccga ttgcagaatg
34201 aatttgctgg aaatatttct gcgctggctg atgcggaaaa tatttcacgt aagattatta
34261 cccgctgtat caacaccgcc aaattgccta aatcagttgt tgctcttttt tctcaccccg
34321 gtgaactatc tgcccggtca ggtgatgcac ttcaaaaagc ctttacagat aaagaggaat
34381 tacttaagca gcaggcatct aaccttcatg agcagaaaaa agctggggtg atatttgaag
34441 ctgaagaagt tatcactctt ttaacttctg tgcttaaaac gtcatctgca tcaagaacta
34501 gtttaagctc acgacatcag tttgctcctg gagcgacagt attgtataag ggcgataaaa
34561 tggtgcttaa cctggacagg tctcgtgttc caactgagtg tatagagaaa attgaggcca
34621 ttcttaagga acttgaaaag ccagcaccct gatgcgacca cgttttagtc tacgtttatc
34681 tgtctttact taatgtcctt tgttacaggc cagaaagcat aactggcctg aatattctct
34741 ctgggcccac tgttccactt gtatcgtcgg tctgataatc agactgggac cacggtccca
34801 ctcgtatcgt cggtctgatt attagtctgg gaccacggtc ccactcgtat cgtcggtctg
34861 attattagtc tgggaccacg gtcccactcg tatcgtcggt ctgataatca gactgggacc
34921 acggtcccac tcgtatcgtc ggtctgatta ttagtctggg accatggtcc cactcgtatc
34981 gtcggtctga ttattagtct gggaccacgg tcccactcgt atcgtcggtc tgattattag
35041 tctggaacca cggtcccact cgtatcgtcg gtctgattat tagtctggga ccacggtccc
35101 actcgtatcg tcggtctgat tattagtctg ggaccacgat cccactcgtg ttgtcggtct
35161 gattatcggt ctgggaccac ggtcccactt gtattgtcga tcagactatc agcgtgagac 35221 tacgattcca tcaatgcctg tcaagggcaa gtattgacat gtcgtcgtaa cctgtagaac 35281 ggagtaacct cggtgtgcgg ttgtatgcct gctgtggatt gctgctgtgt cctgcttatc 35341 cacaacattt tgcgcacggt tatgtggaca aaatacctgg ttacccaggc cgtgccggca 35401 cgttaaccgg gctgcatccg atgcaagtgt gtcgctgtcg acgagctcgc gagctcggac 35461 atgaggttgc cccgtattca gtgtcgctga tttgtattgt ctgaagttgt ttttacgtta 35521 agttgatgca gatcaattaa tacgatacct gcgtcataat tgattatttg acgtggtttg 35581 atggcctcca cgcacgttgt gatatgtaga tgataatcat tatcacttta cgggtccttt 35641 ccggtgatcc gacaggttac ggggcggcga cctcgcgggt tttcgctatt tatgaaaatt 35701 ttccggttta aggcgtttcc gttcttcttc gtcataactt aatgttttta tttaaaatac 35761 cctctgaaaa gaaaggaaac gacaggtgct gaaagcgagc tttttggcct ctgtcgtttc 35821 ctttctctgt ttttgtccgt ggaatgaaca atggaagtcc gagctcatcg ctaataactt 35881 cgtatagcat acattatacg aagttatatt cgat
// pccl-bac-repsars-cov-2_neor-T2A-GLuc.xdna ("GB"), 35689 bp (SEQ ID NO: 3): 1 gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 61 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccacacagat 121 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt cagctgcgca actgttggga 181 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 241 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacTCATGC GTCCATAGTC 301 CCGTTCCGTA ATACGACTCA CTATAgatta aaggtttata ccttcccagg taacaaacca 361 accaactttc gatctcttgt agatctgttc tctaaacgaa ctttaaaatc tgtgtggctg 421 tcactcggct gcatgcttag tgcactcacg cagtataatt aataactaat tactgtcgtt 481 gacaggacac gagtaactcg tctatcttct gcaggctgct tacggtttcg tccgtgttgc 541 agccgatcat cagcacatct aggtttcgtc cgggtgtgac cgaaaggtaa gatggagagc 601 cttgtccctg gtttcaacga gaaaacacac gtccaactca gtttgcctgt tttacaggtt 661 cgcgacgtgc tcgtacgtgg ctttggagac tccgtggagg aggtcttatc agaggcacgt 721 caacatctta aagatggcac ttgtggctta gtagaagttg aaaaaggcgt tttgcctcaa 781 cttgaacagc cctatgtgtt catcaaacgt tcggatgctc gaactgcacc tcatggtcat 841 gttatggttg agctggtagc agaactcgaa ggcattcagt acggtcgtag tggtgagaca 901 cttggtgtcc ttgtccctca tgtgggcgaa ataccagtgg cttaccgcaa ggttcttctt 961 cgtaagaacg gtaataaagg agctggtggc catagttacg gcgccgatct aaagtcattt 1021 gacttaggcg acgagcttgg cactgatcct tatgaagatt ttcaagaaaa ctggaacact 1081 aaacatagca gtggtgttac ccgtgaactc atgcgtgagc ttaacggagg ggcatacact 1141 cgctatgtcg ataacaactt ctgtggccct gatggctacc ctcttgagtg cattaaagac 1201 cttctagcac gtgctggtaa agcttcatgc actttgtccg aacaactgga ctttattgac 1261 actaagaggg gtgtatactg ctgccgtgaa catgagcatg aaattgcttg gtacacggaa 1321 cgttctgaaa agagctatga attgcagaca ccttttgaaa ttaaattggc aaagaaattt 1381 gacaccttca atggggaatg tccaaatttt gtatttccct taaattccat aatcaagact 1441 attcaaccaa gggttgaaaa gaaaaagctt gatggcttta tgggtagaat tcgatctgtc 1501 tatccagttg cgtcaccaaa tgaatgcaac caaatgtgcc tttcaactct catgaagtgt 1561 gatcattgtg gtgaaacttc atggcagacg ggcgattttg ttaaagccac ttgcgaattt 1621 tgtggcactg agaatttgac taaagaaggt gccactactt gtggttactt accccaaaat 1681 gctgttgtta aaatttattg tccagcatgt cacaattcag aagtaggacc tgagcatagt 1741 cttgccgaat accataatga atctggcttg aaaaccattc ttcgtaaggg tggtcgcact 1801 attgcctttg gaggctgtgt gttctcttat gttggttgcc ataacaagtg tgcctattgg 1861 gttccacgtg ctagcgctaa cataggttgt aaccatacag gtgttgttgg agaaggttcc 1921 gaaggtctta atgacaacct tcttgaaata ctccaaaaag agaaagtcaa catcaatatt 1981 gttggtgact ttaaacttaa tgaagagatc gccattattt tggcatcttt ttctgcttcc 2041 acaagtgctt ttgtggaaac tgtgaaaggt ttggattata aagcattcaa acaaattgtt 2101 gaatcctgtg gtaattttaa agttacaaaa ggaaaagcta aaaaaggtgc ctggaatatt 2161 ggtgaacaga aatcaatact gagtcctctt tatgcatttg catcagaggc tgctcgtgtt 2221 gtacgatcaa ttttctcccg cactcttgaa actgctcaaa attctgtgcg tgttttacag 2281 aaggccgcta taacaatact agatggaatt tcacagtatt cactgagact cattgatgct 2341 atgatgttca catctgattt ggctactaac aatctagttg taatggccta cattacaggt 2401 ggtgttgttc agttgacttc gcagtggcta actaacatct ttggcactgt ttatgaaaaa 2461 ctcaaacccg tccttgattg gcttgaagag aagtttaagg aaggtgtaga gtttcttaga 2521 gacggttggg aaattgttaa atttatctca acctgtgctt gtgaaattgt cggtggacaa 2581 attgtcacct gtgcaaagga aattaaggag agtgttcaga cattctttaa gcttgtaaat 2641 aaatttttgg ctttgtgtgc tgactctatc attattggtg gagctaaact taaagccttg 2701 aatttaggtg aaacatttgt cacgcactca aagggattgt acagaaagtg tgttaaatcc 2761 agagaagaaa ctggcctact catgcctcta aaagccccaa aagaaattat cttcttagag 2821 ggagaaacac ttcccacaga agtgttaaca gaggaagttg tcttgaaaac tggtgattta 2881 caaccattag aacaacctac tagtgaagct gttgaagctc cattggttgg tacaccagtt 2941 tgtattaacg ggcttatgtt gctcgaaatc aaagacacag aaaagtactg tgcccttgca 3001 cctaatatga tggtaacaaa caataccttc acactcaaag gcggtgcacc aacaaaggtt 3061 acttttggtg atgacactgt gatagaagtg caaggttaca agagtgtgaa tatcactttt 3121 gaacttgatg aaaggattga taaagtactt aatgagaagt gctctgccta tacagttgaa 3181 ctcggtacag aagtaaatga gttcgcctgt gttgtggcag atgctgtcat aaaaactttg 3241 caaccagtat ctgaattact tacaccactg ggcattgatt tagatgagtg gagtatggct 3301 acatactact tatttgatga gtctggtgag tttaaattgg cttcacatat gtattgttct 3361 ttctaccctc cagatgagga tgaagaagaa ggtgattgtg aagaagaaga gtttgagcca 3421 tcaactcaat atgagtatgg tactgaagat gattaccaag gtaaaccttt ggaatttggt 3481 gccacttctg ctgctcttca acctgaagaa gagcaagaag aagattggtt agatgatgat 3541 agtcaacaaa ctgttggtca acaagacggc agtgaggaca atcagacaac tactattcaa 3601 acaattgttg aggttcaacc tcaattagag atggaactta caccagttgt tcagactatt 3661 gaagtgaata gttttagtgg ttatttaaaa cttactgaca atgtatacat taaaaatgca 3721 gacattgtgg aagaagctaa aaaggtaaaa ccaacagtgg ttgttaatgc agccaatgtt 3781 taccttaaac atggaggagg tgttgcagga gccttaaata aggctactaa caatgccatg 3841 caagttgaat ctgatgatta catagctact aatggaccac ttaaagtggg tggtagttgt 3901 gttttaagcg gacacaatct tgctaaacac tgtcttcatg ttgtcggccc aaatgttaac 3961 aaaggtgaag acattcaact tcttaagagt gcttatgaaa attttaatca gcacgaagtt 4021 ctacttgcac cattattatc agctggtatt tttggtgctg accctataca ttctttaaga 4081 gtttgtgtag atactgttcg cacaaatgtc tacttagctg tctttgataa aaatctctat 4141 gacaaacttg tttcaagctt tttggaaatg aagagtgaaa agcaagttga acaaaagatc 4201 gctgagattc ctaaagagga agttaagcca tttataactg aaagtaaacc ttcagttgaa 4261 cagagaaaac aagatgataa gaaaatcaaa gcttgtgttg aagaagttac aacaactctg 4321 gaagaaacta agttcctcac agaaaacttg ttactttata ttgacattaa tggcaatctt 4381 catccagatt ctgccactct tgttagtgac attgacatca ctttcttaaa gaaagatgct 4441 ccatatatag tgggtgatgt tgttcaagag ggtgttttaa ctgctgtggt tatacctact 4501 aaaaaggctg gtggcactac tgaaatgcta gcgaaagctt tgagaaaagt gccaacagac 4561 aattatataa ccacttaccc gggtcagggt ttaaatggtt acactgtaga ggaggcaaag 4621 acagtgctta aaaagtgtaa aagtgccttt tacattctac catctattat ctctaatgag 4681 aagcaagaaa ttcttggaac tgtttcttgg aatttgcgag aaatgcttgc acatgcagaa 4741 gaaacacgca aattaatgcc tgtctgtgtg gaaactaaag ccatagtttc aactatacag 4801 cgtaaatata agggtattaa aatacaagag ggtgtggttg attatggtgc tagattttac 4861 ttttacacca gtaaaacaac tgtagcgtca cttatcaaca cacttaacga tctaaatgaa 4921 actcttgtta caatgccact tggctatgta acacatggct taaatttgga agaagctgct 4981 cggtatatga gatctctcaa agtgccagct acagtttctg tttcttcacc tgatgctgtt 5041 acagcgtata atggttatct tacttcttct tctaaaacac ctgaagaaca ttttattgaa 5101 accatctcac ttgctggttc ctataaagat tggtcctatt ctggacaatc tacacaacta 5161 ggtatagaat ttcttaagag aggtgataaa agtgtatatt acactagtaa tcctaccaca 5221 ttccacctag atggtgaagt tatcaccttt gacaatctta agacacttct ttctttgaga 5281 gaagtgagga ctattaaggt gtttacaaca gtagacaaca ttaacctcca cacgcaagtt 5341 gtggacatgt caatgacata tggacaacag tttggtccaa cttatttgga tggagctgat 5401 gttactaaaa taaaacctca taattcacat gaaggtaaaa cattttatgt tttacctaat 5461 gatgacactc tacgtgttga ggcttttgag tactaccaca caactgatcc tagttttctg 5521 ggtaggtaca tgtcagcatt aaatcacact aaaaagtgga aatacccaca agttaatggt 5581 ttaacttcta ttaaatgggc agataacaac tgttatcttg ccactgcatt gttaacactc 5641 caacaaatag agttgaagtt taatccacct gctctacaag atgcttatta cagagcaagg 5701 gctggtgaag ctgctaactt ttgtgcactt atcttagcct actgtaataa gacagtaggt 5761 gagttaggtg atgttagaga aacaatgagt tacttgtttc aacatgccaa tttagattct 5821 tgcaaaagag tcttgaacgt ggtgtgtaaa acttgtggac aacagcagac aacccttaag 5881 ggtgtagaag ctgttatgta catgggcaca ctttcttatg aacaatttaa gaaaggtgtt 5941 cagatacctt gtacgtgtgg taaacaagct acaaaatatc tagtacaaca ggagtcacct 6001 tttgttatga tgtcagcacc acctgctcag tatgaactta agcatggtac atttacttgt 6061 gctagtgagt acactggtaa ttaccagtgt ggtcactata aacatataac ttctaaagaa 6121 actttgtatt gcatagacgg tgctttactt acaaagtcct cagaatacaa aggtcctatt 6181 acggatgttt tctacaaaga aaacagttac acaacaacca taaaaccagt tacttataaa 6241 ttggatggtg ttgtttgtac agaaattgac cctaagttgg acaattatta taagaaagac 6301 aattcttatt tcacagagca accaattgat cttgtaccaa accaaccata tccaaacgca 6361 agcttcgata attttaagtt tgtatgtgat aatatcaaat ttgctgatga tttaaaccag 6421 ttaactggtt ataagaaacc tgcttcaaga gagcttaaag ttacattttt ccctgactta 6481 aatggtgatg tggtggctat tgattataaa cactacacac cctcttttaa gaaaggagct 6541 aaattgttac ataaacctat tgtttggcat gttaacaatg caactaataa agccacgtat 6601 aaaccaaata cctggtgtat acgttgtctt tggagcacaa aaccagttga aacatcaaat 6661 tcgtttgatg tactgaagtc agaggacgcg cagggaatgg ataatcttgc ctgcgaagat 6721 ctaaaaccag tctctgaaga agtagtggaa aatcctacca tacagaaaga cgttcttgag 6781 tgtaatgtga aaactaccga agttgtagga gacattatac ttaaaccagc aaataatagt 6841 ttaaaaatta cagaagaggt tggccacaca gatctaatgg ctgcttatgt agacaattct 6901 agtcttacta ttaagaaacc taatgaatta tctagagtat taggtttgaa aacccttgct 6961 actcatggtt tagctgctgt taatagtgtc ccttgggata ctatagctaa ttatgctaag 7021 ccttttctta acaaagttgt tagtacaact actaacatag ttacacggtg tttaaaccgt 7081 gtttgtacta attatatgcc ttatttcttt actttattgc tacaattgtg tacttttact 7141 agaagtacaa attctagaat taaagcatct atgccgacta ctatagcaaa gaatactgtt 7201 aagagtgtcg gtaaattttg tctagaggct tcatttaatt atttgaagtc acctaatttt 7261 tctaaactga t33^t3tt31 aatttggttt ttactattaa gtgtttgcct aggttcttta 7321 atctactcaa ccgctgcttt aggtgtttta atgtctaatt taggcatgcc ttcttactgt 7381 actggttaca gagaaggcta tttgaactct actaatgtca ctattgcaac ctactgtact 7441 ggttctatac cttgtagtgt ttgtcttagt ggtttagatt ctttagacac ctatccttct 7501 ttagaaacta tacaaattac catttcatct tttaaatggg atttaactgc ttttggctta 7561 gttgcagagt ggtttttggc atatattctt ttcactaggt ttttctatgt acttggattg 7621 gctgcaatca tgcaattgtt tttcagctat tttgcagtac attttattag taattcttgg 7681 cttatgtggt taataattaa tcttgtacaa atggccccga tttcagctat ggttagaatg 7741 tacatcttct ttgcatcatt ttattatgta tggaaaagtt atgtgcatgt tgtagacggt 7801 tgtaattcat caacttgtat gatgtgttac aaacgtaata gagcaacaag agtcgaatgt 7861 acaactattg ttaatggtgt tagaaggtcc ttttatgtct atgctaatgg aggtaaaggc 7921 ttttgcaaac tacacaattg gaattgtgtt aattgtgata cattctgtgc tggtagtaca 7981 tttattagtg atgaagttgc gagagacttg tcactacagt ttaaaagacc aataaatcct 8041 actgaccagt cttcttacat cgttgatagt gttacagtga agaatggttc catccatctt 8101 tactttgata aagctggtca aaagacttat gaaagacatt ctctctctca ttttgttaac 8161 ttagacaacc tgagagctaa taacactaaa ggttcattgc ctattaatgt tatagttttt 8221 gatggtaaat caaaatgtga agaatcatct gcaaaatcag cgtctgttta ctacagtcag 8281 cttatgtgtc aacctatact gttactagat caggcattag tgtctgatgt tggtgatagt 8341 gcggaagttg cagttaaaat gtttgatgct tacgttaata cgttttcatc aacttttaac 8401 gtaccaatgg aaaaactcaa aacactagtt gcaactgcag aagctgaact tgcaaagaat 8461 gtgtccttag acaatgtctt atctactttt atttcagcag ctcggcaagg gtttgttgat 8521 tcagatgtag aaactaaaga tgttgttgaa tgtcttaaat tgtcacatca atctgacata 8581 gaagttactg gcgatagttg taataactat atgctcacct ataacaaagt tgaaaacatg 8641 acaccccgtg accttggtgc ttgtattgac tgtagtgcgc gtcatattaa tgcgcaggta 8701 gcaaaaagtc acaacattgc tttgatatgg aacgttaaag atttcatgtc attgtctgaa 8761 caactacgaa aacaaatacg tagtgctgct ^33^3CC^^13 acttaccttt taagttgaca 8821 tgtgcaacta ctagacaagt tgttaatgtt gtaacaacaa agatagcact taagggtggt 8881 aaaattgtta ataattggtt gaagcagtta attaaagtta cacttgtgtt cctttttgtt 8941 gctgctattt tctatttaat aacacctgtt catgtcatgt ctaaacatac tgacttttca 9001 agtgaaatca taggatacaa ggctattgat ggtggtgtca ctcgtgacat agcatctaca 9061 gatacttgtt ttgctaacaa acatgctgat tttgacacat ggtttagcca gcgtggtggt 9121 agttatacta atgacaaagc ttgcccattg attgctgcag tcataacaag agaagtgggt 9181 tttgtcgtgc ctggtttgcc tggcacgata ttacgcacaa ctaatggtga ctttttgcat 9241 ttcttaccta gagtttttag tgcagttggt aacatctgtt acacaccatc aaaacttata 9301 gagtacactg actttgcaac atcagcttgt gttttggctg ctgaatgtac aatttttaaa 9361 gatgcttctg gtaagccagt accatattgt tatgatacca atgtactaga aggttctgtt
9421 gcttatgaaa gtttacgccc tgacacacgt tatgtgctca tggatggctc tattattcaa
9481 tttcctaaca cctaccttga aggttctgtt agagtggtaa caacttttga ttctgagtac
9541 tgtaggcacg gcacttgtga aagatcagaa gctggtgttt gtgtatctac tagtggtaga
9601 tgggtactta acaatgatta ttacagatct ttaccaggag ttttctgtgg tgtagatgct
9661 gtaaatttac ttactaatat gtttacacca ctaattcaac ctattggtgc tttggacata
9721 tcagcatcta tagtagctgg tggtattgta gctatcgtag taacatgcct tgcctactat
9781 tttatgaggt ttagaagagc ttttggtgaa tacagtcatg tagttgcctt taatacttta
9841 ctattcctta tgtcattcac tgtactctgt ttaacaccag tttactcatt cttacctggt
9901 gtttattctg ttatttactt gtacttgaca ttttatctta ctaatgatgt ttctttttta
9961 gcacatattc agtggatggt tatgttcaca cctttagtac ctttctggat aacaattgct
10021 tatatcattt gtatttccac aaagcatttc tattggttct ttagtaatta cctaaagaga
10081 cgtgtagtct ttaatggtgt ttcctttagt acttttgaag aagctgcgct gtgcaccttt
10141 ttgttaaata aagaaatgta tctaaagttg cgtagtgatg tgctattacc tcttacgcaa
10201 tataatagat acttagctct ttataataag tacaagtatt ttagtggagc aatggataca
10261 actagctaca gagaagctgc ttgttgtcat ctcgcaaagg ctctcaatga cttcagtaac
10321 tcaggttctg atgttcttta ccaaccacca caaacctcta tcacctcagc tgttttgcag
10381 agtggtttta gaaaaatggc attcccatct ggtaaagttg agggttgtat ggtacaagta
10441 acttgtggta caactacact taacggtctt tggcttgatg acgtagttta ctgtccaaga
10501 catgtgatct gcacctctga agacatgctt aaccctaatt atgaagattt actcattcgt
10561 aagtctaatc ataatttctt ggtacaggct ggtaatgttc aactcagggt tattggacat
10621 tctatgcaaa attgtgtact taagcttaag gttgatacag ccaatcctaa gacacctaag
10681 tataagtttg ttcgcattca accaggacag actttttcag tgttagcttg ttacaatggt
10741 tcaccatctg gtgtttacca atgtgctatg aggcccaatt tcactattaa gggttcattc
10801 cttaatggtt catgtggtag tgttggtttt aacatagatt atgactgtgt ctctttttgt
10861 tacatgcacc atatggaatt accaactgga gttcatgctg gcacagactt agaaggtaac
10921 ttttatggac cttttgttga caggcaaaca gcacaagcag ctggtacgga cacaactatt
10981 acagttaatg ttttagcttg gttgtacgct gctgttataa atggagacag gtggtttctc
11041 aatcgattta ccacaactct taatgacttt aaccttgtgg ctatgaagta caattatgaa
11101 cctctaacac aagaccatgt tgacatacta ggacctcttt ctgctcaaac tggaattgcc
11161 gttttagata tgtgtgcttc attaaaagaa ttactgcaaa atggtatgaa tggacgtacc
11221 atattgggta gtgctttatt agaagatgaa tttacacctt ttgatgttgt tagacaatgc
11281 tcaggtgtta ctttccaaag tgcagtgaaa agaacaatca agggtacaca ccactggttg
11341 ttactcacaa ttttgacttc acttttagtt ttagtccaga gtactcaatg gtctttgttc
11401 ttttttttgt atgaaaatgc ctttttacct tttgctatgg gtattattgc tatgtctgct
11461 tttgcaatga tgtttgtcaa acataagcat gcatttctct gtttgttttt gttaccttct
11521 cttgccactg tagcttattt taatatggtc tatatgcctg ctagttgggt gatgcgtatt
11581 atgacatggt tggatatggt tgatactagt ttgtctggtt ttaagctaaa agactgtgtt
11641 atgtatgcat cagctgtagt gttactaatc cttatgacag caagaactgt gtatgatgat
11701 ggtgctagga gagtgtggac acttatgaat gtcttgacac tcgtttataa agtttattat
11761 ggtaatgctt tagatcaagc catttccatg tgggctctta taatctctgt tacttctaac
11821 tactcaggtg tagttacaac tgtcatgttt ttggccagag gtattgtttt tatgtgtgtt
11881 gagtattgcc ctattttctt cataactggt aatacacttc agtgtataat gctagtttat
11941 tgtttcttag gctatttttg tacttgttac tttggcctct tttgtttact caaccgctac
12001 tttagactga ctcttggtgt ttatgattac ttagtttcta cacaggagtt tagatatatg
12061 aattcacagg gactactccc acccaagaat agcatagatg ccttcaaact caacattaaa
12121 ttgttgggtg ttggtggcaa accttgtatc aaagtagcca ctgtacagtc taaaatgtca
12181 gatgtaaagt gcacatcagt agtcttactc tcagttttgc aacaactcag agtagaatca
12241 tcatctaaat tgtgggctca atgtgtccag ttacacaatg acattctctt agctaaagat
12301 actactgaag cctttgaaaa aatggtttca ctactttctg ttttgctttc catgcagggt
12361 gctgtagaca taaacaagct ttgtgaagaa atgctggaca acagggcaac cttacaagct
12421 atagcctcag agtttagttc ccttccatca tatgcagctt ttgctactgc tcaagaagct
12481 tatgagcagg ctgttgctaa tggtgattct gaagttgttc ttaaaaagtt gaagaagtct
12541 ttgaatgtgg ctaaatctga atttgaccgt gatgcagcca tgcaacgtaa gttggaaaag
12601 atggctgatc aagctatgac ccaaatgtat aaacaggcta gatctgagga caagagggca
12661 aaagttacta gtgctatgca gacaatgctt ttcactatgc ttagaaagtt ggataatgat
12721 gcactcaaca acattatcaa caatgcaaga gatggttgtg ttcccttgaa cataatacct 12781 cttacaacag cagccaaact aatggttgtc ataccagact ataacacata taaaaatacg
12841 tgtgatggta caacatttac ttatgcatca gcattgtggg aaatccaaca ggttgtagat
12901 gcagatagta aaattgttca acttagtgaa attagtatgg acaattcacc taatttagca
12961 tggcctctta ttgtaacagc tttaagggcc aattctgctg tcaaattaca gaataatgag
13021 cttagtcctg ttgcactacg acagatgtct tgtgctgccg gtactacaca aactgcttgc
13081 actgatgaca atgcgttagc ttactacaac acaacaaagg gaggtaggtt tgtacttgca
13141 ctgttatccg atttacagga tttgaaatgg gctagattcc ctaagagtga tggaactggt
13201 actatctata cagaactgga accaccttgt aggtttgtta cagacacacc taaaggtcct
13261 aaagtgaagt atttatactt tattaaagga ttaaacaacc taaatagagg tatggtactt
13321 ggtagtttag ctgccacagt acgtctacaa gctggtaatg caacagaagt gcctgccaat
13381 tcaactgtat tatctttctg tgcttttgct gtagatgctg ctaaagctta caaagattat
13441 ctagctagtg ggggacaacc aatcactaat tgtgttaaga tgttgtgtac acacactggt
13501 actggtcagg caataacagt tacaccggaa gccaatatgg atcaagaatc ctttggtggt
13561 gcatcgtgtt gtctgtactg ccgttgccac atagatcatc caaatcctaa aggattttgt
13621 gacttaaaag gtaagtatgt acaaatacct acaacttgtg ctaatgaccc tgtgggtttt
13681 acacttaaaa acacagtctg taccgtctgc ggtatgtgga aaggttatgg ctgtagttgt
13741 gatcaactcc gcgaacccat gcttcagtca gctgatgcac aatcgttttt aaacgggttt
13801 gcggtgtaag tgcagcccgt cttacaccgt gcggcacagg cactagtact gatgtcgtat
13861 acagggcttt tgacatctac aatgataaag tagctggttt tgctaaattc ctaaaaacta
13921 attgttgtcg cttccaagaa aaggacgaag atgacaattt aattgattct tactttgtag
13981 ttaagagaca cactttctct aactaccaac atgaagaaac aatttataat ttacttaagg
14041 attgtccagc tgttgctaaa catgacttct ttaagtttag aatagacggt gacatggtac
14101 cacatatatc acgtcaacgt cttactaaat acacaatggc agacctcgtc tatgctttaa
14161 ggcattttga tgaaggtaat tgtgacacat taaaagaaat acttgtcaca tacaattgtt
14221 gtgatgatga ttatttcaat aaaaaggact ggtatgattt tgtagaaaac ccagatatat
14281 tacgcgtata cgccaactta ggtgaacgtg tacgccaagc tttgttaaaa acagtacaat
14341 tctgtgatgc catgcgaaat gctggtattg ttggtgtact gacattagat aatcaagatc
14401 tcaatggtaa ctggtatgat ttcggtgatt tcatacaaac cacgccaggt agtggagttc
14461 ctgttgtaga ttcttattat tcattgttaa tgcctatatt aaccttgacc agggctttaa
14521 ctgcagagtc acatgttgac actgacttaa caaagcctta cattaagtgg gatttgttaa
14581 aatatgactt cacggaagag aggttaaaac tctttgaccg ttattttaaa tattgggatc
14641 agacatacca cccaaattgt gttaactgtt tggatgacag atgcattctg cattgtgcaa
14701 actttaatgt tttattctct acagtgttcc cacctacaag ttttggacca ctagtgagaa
14761 aaatatttgt tgatggtgtt ccatttgtag tttcaactgg ataccacttc agagagctag
14821 gtgttgtaca taatcaggat gtaaacttac atagctctag acttagtttt aaggaattac
14881 ttgtgtatgc tgctgaccct gctatgcacg ctgcttctgg taatctatta ctagataaac
14941 gcactacgtg cttttcagta gctgcactta ctaacaatgt tgcttttcaa actgtcaaac
15001 ccggtaattt taacaaagac ttctatgact ttgctgtgtc taagggtttc tttaaggaag
15061 gaagttctgt tgaattaaaa cacttcttct ttgctcagga tggtaatgct gctatcagcg
15121 attatgacta ctatcgttat aatctaccaa caatgtgtga tatcagacaa ctactatttg
15181 tagttgaagt tgttgataag tactttgatt gttacgatgg tggctgtatt aatgctaacc
15241 aagtcatcgt caacaaccta gacaaatcag ctggttttcc atttaataaa tggggtaagg
15301 ctagacttta ttatgattca atgagttatg aggatcaaga tgcacttttc gcatatacaa
15361 aacgtaatgt catccctact ataactcaaa tgaatcttaa gtatgccatt agtgcaaaga
15421 atagagctcg caccgtagct ggtgtctcta tctgtagtac tatgaccaat agacagtttc
15481 atcaaaaatt attgaaatca atagccgcca ctagaggagc tactgtagta attggaacaa
15541 gcaaattcta tggtggttgg cacaacatgt taaaaactgt ttatagtgat gtagaaaacc
15601 ctcaccttat gggttgggat tatcctaaat gtgatagagc catgcctaac atgcttagaa
15661 ttatggcctc acttgttctt gctcgcaaac atacaacgtg ttgtagcttg tcacaccgtt
15721 tctatagatt agctaatgag tgtgctcaag tattgagtga aatggtcatg tgtggcggtt
15781 cactatatgt taaaccaggt ggaacctcat caggagatgc cacaactgct tatgctaata
15841 gtgtttttaa catttgtcaa gctgtcacgg ccaatgttaa tgcactttta tctactgatg
15901 gtaacaaaat tgccgataag tatgtccgca atttacaaca cagactttat gagtgtctct
15961 atagaaatag agatgttgac acagactttg tgaatgagtt ttacgcatat ttgcgtaaac
16021 atttctcaat gatgatactc tctgacgatg ctgttgtgtg tttcaatagc acttatgcat
16081 ctcaaggtct agtggctagc ataaagaact ttaagtcagt tctttattat caaaacaatg
16141 tttttatgtc tgaagcaaaa tgttggactg agactgacct tactaaagga cctcatgaat 16201 tttgctctca acatacaatg ctagttaaac agggtgatga ttatgtgtac cttccttacc
16261 cagatccatc aagaatccta ggggccggct gttttgtaga tgatatcgta aaaacagatg
16321 gtacacttat gattgaacgg ttcgtgtctt tagctataga tgcttaccca cttactaaac
16381 atcctaatca ggagtatgct gatgtctttc atttgtactt acaatacata agaaagctac
16441 atgatgagtt aacaggacac atgttagaca tgtattctgt tatgcttact aatgataaca
16501 cttcaaggta ttgggaacct gagttttatg aggctatgta cacaccgcat acagtcttac
16561 aggctgttgg ggcttgtgtt ctttgcaatt cacagacttc attaagatgt ggtgcttgca
16621 tacgtagacc attcttatgt tgtaaatgct gttacgacca tgtcatatca acatcacata
16681 aattagtctt gtctgttaat ccgtatgttt gcaatgctcc aggttgtgat gtcacagatg
16741 tgactcaact ttacttagga ggtatgagct attattgtaa atcacataaa ccacccatta
16801 gttttccatt gtgtgctaat ggacaagttt ttggtttata taaaaataca tgtgttggta
16861 gcgataatgt tactgacttt aatgcaattg caacatgtga ctggacaaat gctggtgatt
16921 acattttagc taacacctgt actgaaagac tcaagctttt tgcagcagaa acgctcaaag
16981 ctactgagga gacatttaaa ctgtcttatg gtattgctac tgtacgtgaa gtgctgtctg
17041 acagagaatt acatctttca tgggaagttg gtaaacctag accaccactt aaccgaaatt
17101 atgtctttac tggttatcgt gtaactaaaa acagtaaagt acaaatagga gagtacacct
17161 ttgaaaaagg tgactatggt gatgctgttg tttaccgagg tacaacaact tacaaattaa
17221 atgttggtga ttattttgtg ctgacatcac atacagtaat gccattaagt gcacctacac
17281 tagtgccaca agagcactat gttagaatta ctggcttata cccaacactc aatatctcag
17341 atgagttttc tagcaatgtt gcaaattatc aaaaggttgg tatgcaaaag tattctacac
17401 tccagggacc acctggtact ggtaagagtc attttgctat tggcctagct ctctactacc
17461 cttctgctcg catagtgtat acagcttgct ctcatgccgc tgttgatgca ctatgtgaga
17521 aggcattaaa atatttgcct atagataaat gtagtagaat tatacctgca cgtgctcgtg
17581 tagagtgttt tgataaattc aaagtgaatt caacattaga acagtatgtc ttttgtactg
17641 taaatgcatt gcctgagacg acagcagata tagttgtctt tgatgaaatt tcaatggcca
17701 caaattatga tttgagtgtt gtcaatgcca gattacgtgc taagcactat gtgtacattg
17761 gcgaccctgc tcaattacct gcaccacgca cattgctaac taagggcaca ctagaaccag
17821 aatatttcaa ttcagtgtgt agacttatga aaactatagg tccagacatg ttcctcggaa
17881 cttgtcggcg ttgtcctgct gaaattgttg acactgtgag tgctttggtt tatgataata
17941 agcttaaagc acataaagac aaatcagctc aatgctttaa aatgttttat aagggtgtta
18001 tcacgcatga tgtttcatct gcaattaaca ggccacaaat aggcgtggta agagaattcc
18061 ttacacgtaa ccctgcttgg agaaaagctg tctttatttc accttataat tcacagaatg
18121 ctgtagcctc aaagattttg ggactaccaa ctcaaactgt tgattcatca cagggctcag
18181 aatatgacta tgtcatattc actcaaacca ctgaaacagc tcactcttgt aatgtaaaca
18241 gatttaatgt tgctattacc agagcaaaag taggcatact ttgcataatg tctgatagag
18301 acctttatga caagttgcaa tttacaagtc ttgaaattcc acgtaggaat gtggcaactt
18361 tacaagctga aaatgtaaca ggactcttta aagattgtag taaggtaatc actgggttac
18421 atcctacaca ggcacctaca cacctcagtg ttgacactaa attcaaaact gaaggtttat
18481 gtgttgacat acctggcata cctaaggaca tgacctatag aagactcatc tctatgatgg
18541 gttttaaaat gaattatcaa gttaatggtt accctaacat gtttatcacc cgcgaagaag
18601 ctataagaca tgtacgtgca tggattggct tcgatgtcga ggggtgtcat gctactagag
18661 aagctgttgg taccaattta cctttacagc taggtttttc tacaggtgtt aacctagttg
18721 ctgtacctac aggttatgtt gatacaccta ataatacaga tttttccaga gttagtgcta
18781 aaccaccgcc tggagatcaa tttaaacacc tcataccact tatgtacaaa ggacttcctt
18841 ggaatgtagt gcgtataaag attgtacaaa tgttaagtga cacacttaaa aatctctctg
18901 acagagtcgt atttgtctta tgggcacatg gctttgagtt gacatctatg aagtattttg
18961 tgaaaatagg acctgagcgc acctgttgtc tatgtgatag acgtgccaca tgcttttcca
19021 ctgcttcaga cacttatgcc tgttggcatc attctattgg atttgattac gtctataatc
19081 cgtttatgat tgatgttcaa caatggggtt ttacaggtaa cctacaaagc aaccatgatc
19141 tgtattgtca agtccatggt aatgcacatg tagctagttg tgatgcaatc atgactaggt
19201 gtctagctgt ccacgagtgc tttgttaagc gtgttgactg gactattgaa tatcctataa
19261 ttggtgatga actgaagatt aatgcggctt gtagaaaggt tcaacacatg gttgttaaag
19321 ctgcattatt agcagacaaa ttcccagttc ttcacgacat tggtaaccct aaagctatta
19381 agtgtgtacc tcaagctgat gtagaatgga agttctatga tgcacagcct tgtagtgaca
19441 aagcttataa aatagaagaa ttattctatt cttatgccac acattctgac aaattcacag
19501 atggtgtatg cctattttgg aattgcaatg tcgatagata tcctgctaat tccattgttt
19561 gtagatttga cactagagtg ctatctaacc ttaacttgcc tggttgtgat ggtggcagtt 19621 tgtatgtaaa taaacatgca ttccacacac cagcttttga taaaagtgct tttgttaatt
19681 taaaacaatt accatttttc tattactctg acagtccatg tgagtctcat ggaaaacaag
19741 tagtgtcaga tatagattat gtaccactaa agtctgctac gtgtataaca cgttgcaatt
19801 taggtggtgc tgtctgtaga catcatgcta atgagtacag attgtatctc gatgcttata
19861 acatgatgat ctcagctggc tttagcttgt gggtttacaa acaatttgat acttataacc
19921 tctggaacac ttttacaaga cttcagagtt tagaaaatgt ggcttttaat gttgtaaata
19981 agggacactt tgatggacaa cagggtgaag taccagtttc tatcattaat aacactgttt
20041 acacaaaagt tgatggtgtt gatgtagaat tgtttgaaaa taaaacaaca ttacctgtta
20101 atgtagcatt tgagctttgg gctaagcgca acattaaacc agtaccagag gtgaaaatac
20161 tcaataattt gggtgtggac attgctgcta atactgtgat ctgggactac aaaagagatg
20221 ctccagcaca tatatctact attggtgttt gttctatgac tgacatagcc aagaaaccaa
20281 ctgaaacgat ttgtgcacca ctcactgtct tttttgatgg tagagttgat ggtcaagtag
20341 acttatttag aaatgcccgt aatggtgttc ttattacaga aggtagtgtt aaaggtttac
20401 aaccatctgt aggtcccaaa caagctagtc ttaatggagt cacattaatt ggagaagccg
20461 taaaaacaca gttcaattat tataagaaag ttgatggtgt tgtccaacaa ttacctgaaa
20521 cttactttac tcagagtaga aatttacaag aatttaaacc caggagtcaa atggaaattg
20581 atttcttaga attagctatg gatgaattca ttgaacggta taaattagaa ggctatgcct
20641 tcgaacatat cgtttatgga gattttagtc atagtcagtt aggtggttta catctactga
20701 ttggactagc taaacgtttt aaggaatcac cttttgaatt agaagatttt attcctatgg
20761 acagtacagt taaaaactat ttcataacag atgcgcaaac aggttcatct aagtgtgtgt
20821 gttctgttat tgatttatta cttgatgatt ttgttgaaat aataaaatcc caagatttat
20881 ctgtagtttc taaggttgtc aaagtgacta ttgactatac agaaatttca tttatgcttt
20941 ggtgtaaaga tggccatgta gaaacatttt acccaaaatt acaatctagt caagcgtggc
21001 aaccgggtgt tgctatgcct aatctttaca aaatgcaaag aatgctatta gaaaagtgtg
21061 accttcaaaa ttatggtgat agtgcaacat tacctaaagg cataatgatg aatgtcgcaa
21121 aatatactca actgtgtcaa tatttaaaca cattaacatt agctgtaccc tataatatga
21181 gagttataca ttttggtgct ggttctgata aaggagttgc accaggtaca gctgttttaa
21241 gacagtggtt gcctacgggt acgctgcttg tcgattcaga tcttaatgac tttgtctctg
21301 atgcagattc aactttgatt ggtgattgtg caactgtaca tacagctaat aaatgggatc
21361 tcattattag tgatatgtac gaccctaaga ctaaaaatgt tacaaaagaa aatgactcta
21421 aagagggttt tttcacttac atttgtgggt ttatacaaca aaagctagct cttggaggtt
21481 ccgtggctat aaagataaca gaacattctt ggaatgctga tctttataag ctcatgggac
21541 acttcgcatg gtggacagcc tttgttacta atgtgaatgc gtcatcatct gaagcatttt
21601 taattggatg taattatctt ggcaaaccac gcgaacaaat agatggttat gtcatgcatg
21661 caaattacat attttggagg aatacaaatc caattcagtt gtcttcctat tctttatttg
21721 acatgagtaa atttcccctt aaattaaggg gtactgctgt tatgtcttta aaagaaggtc
21781 aaatcaatga tatgatttta tctcttctta gtaaaggtag acttataatt agagaaaaca
21841 acagagttgt tatttctagt gatgttcttg ttaacaacta aacgaacaAT GGTTATTGAA
21901 CAAGATGGAT TGCACGCAGG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC
21961 TGGGCACAAC AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG
22021 CGCCCGGTTC TTTTTGTCAA GACCGACCTG TCCGGTGCCC TGAATGAACT GCAGGACGAG
22081 GCAGCGCGGC TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT
22141 GTCACTGAAG CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG
22201 TCATCTCACC TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG
22261 CATACGCTTG ATCCGGCTAC CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA
22321 GCACGTACTC GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG
22381 GGGCTCGCGC CAGCCGAACT GTTCGCCAGG CTCAAGGCGC GCATGCCCGA CGGCGAGGAT
22441 CTCGTCGTGA CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT
22501 TCTGGATTCA TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG
22561 GCTACCCGTG ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT
22621 TACGGTATCG CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC
22681 TTCGAGGGCA GAGGAAGTCT TCTAACATGC GGTGACGTGG AGGAGAATCC CGGCCCTatg
22741 ggagtcaaag ttctgtttgc cctgatctgc atcgctgtgg ccgaggccaa gcccaccgag
22801 aacaacgaag acttcaacat cgtggccgtg gccagcaact tcgcgaccac ggatctcgat
22861 gctgaccgcg ggaagttgcc cggcaagaag ctgccgctgg aggtgctcaa agagatggaa
22921 gccaatgccc ggaaagctgg ctgcaccagg ggctgtctga tctgcctgtc ccacatcaag
22981 tgcacgccca agatgaagaa gttcatccca ggacgctgcc acacctacga aggcgacaaa 23041 gagtccgcac agggcggcat aggcgaggcg atcgtcgaca ttcctgagat tcctgggttc
23101 aaggacttgg agcccatgga gcagttcatc gcacaggtcg atctgtgtgt ggactgcaca
23161 actggctgcc tcaaagggct tgccaacgtg cagtgttctg acctgctcaa gaagtggctg
23221 ccgcaacgct gtgcgacctt tgccagcaag atccagggcc aggtggacaa gatcaagggg
23281 gccggtggtg acTAAacgaa cttatggatt tgtttatgag aatcttcaca attggaactg
23341 taactttgaa gcaaggtgaa atcaaggatg ctactccttc agattttgtt cgcgctactg
23401 caacgatacc gatacaagcc tcactccctt tcggatggct tattgttggc gttgcacttc
23461 ttgctgtttt tcagagcgct tccaaaatca taaccctcaa aaagagatgg caactagcac
23521 tctccaaggg tgttcacttt gtttgcaact tgctgttgtt gtttgtaaca gtttactcac
23581 accttttgct cgttgctgct ggccttgaag ccccttttct ctatctttat gctttagtct
23641 acttcttgca gagtataaac tttgtaagaa taataatgag gctttggctt tgctggaaat
23701 gccgttccaa aaacccatta ctttatgatg ccaactattt tctttgctgg catactaatt
23761 gttacgacta ttgtatacct tacaatagtg taacttcttc aattgtcatt acttcaggtg
23821 atggcacaac aagtcctatt tctgaacatg actaccagat tggtggttat actgaaaaat
23881 gggaatctgg agtaaaagac tgtgttgtat tacacagtta cttcacttca gactattacc
23941 agctgtactc aactcaattg agtacagaca ctggtgttga acatgttacc ttcttcatct
24001 acaataaaat tgttgatgag cctgaagaac atgtccaaat tcacacaatc gacggttcat
24061 ccggagttgt taatccagta atggaaccaa tttatgatga accgacgacg actactagcg
24121 tgcctttgta agcacaagct gatgagtacg aacttatgta ctcattcgtt tcggaagaga
24181 caggtacgtt aatagttaat agcgtacttc tttttcttgc tttcgtggta ttcttgctag
24241 ttacactagc catccttact gcgcttcgat tgtgtgcgta ctgctgcaat attgttaacg
24301 tgagtcttgt aaaaccttct ttttacgttt actctcgtgt taaaaatctg aattcttcta
24361 gagttcctga tcttctggtc taaacgaact ^3313Lt^L3 ttagtttttc tgtttggaac
24421 tttaatttta gccatggcag attccaacgg tactattacc gttgaagagc ttaaaaagct
24481 ccttgaacaa tggaacctag taataggttt cctattcctt acatggattt gtcttctaca
24541 atttgcctat gccaacagga ataggttttt gtatataatt aagttaattt tcctctggct
24601 gttatggcca gtaactttag cttgttttgt gcttgctgct gtttacagaa taaattggat
24661 caccggtgga attgctatcg caatggcttg tcttgtaggc ttgatgtggc tcagctactt
24721 cattgcttct ttcagactgt ttgcgcgtac gcgttccatg tggtcattca atccagaaac
24781 taacattctt ctcaacgtgc cactccatgg cactattctg accagaccgc ttctagaaag
24841 tgaactcgta atcggagctg tgatccttcg tggacatctt cgtattgctg gacaccatct
24901 aggacgctgt gacatcaagg acctgcctaa agaaatcact gttgctacat cacgaacgct
24961 ttcttattac aaattgggag cttcgcagcg tgtagcaggt gactcaggtt ttgctgcata
25021 cagtcgctac aggattggca actataaatt aaacacagac cattccagta gcagtgacaa
25081 tattgctttg cttgtacagt aagtgacaac agatgtttca tctcgttgac tttcaggtta
25141 ctatagcaga gatattacta attattatga ggacttttaa agtttccatt tggaatcttg
25201 attacatcat aaacctcata attaaaaatt tatctaagtc actaactgag aataaatatt
25261 ctcaattaga tgaagagcaa ccaatggaga ttgattaaac gaacatgaaa attattcttt
25321 tcttggcact gataacactc gctacttgtg agctttatca ctaccaagag tgtgttagag
25381 gtacaacagt acttttaaaa gaaccttgct cttctggaac atacgagggc aattcaccat
25441 ttcatcctct agctgataac aaatttgcac tgacttgctt tagcactcaa tttgcttttg
25501 cttgtcctga cggcgtaaaa cacgtctatc agttacgtgc cagatcagtt tcacctaaac
25561 tgttcatcag acaagaggaa gttcaagaac tttactctcc aatttttctt attgttgcgg
25621 caatagtgtt tataacactt tgcttcacac tcaaaagaaa gacagaatga ttgaactttc
25681 attaattgac ttctatttgt gctttttagc ctttctgcta ttccttgttt taattatgct
25741 tattatcttt tggttctcac ttgaactgca agatcataat gaaacttgtc acgcctaaac
25801 gaacatgaaa tttcttgttt tcttaggaat catcacaact gtagctgcat ttcaccaaga
25861 atgtagttta cagtcatgta ctcaacatca accatatgta gttgatgacc cgtgtcctat
25921 tcacttctat tctaaatggt atattagagt aggagctaga aaatcagcac ctttaattga
25981 attgtgcgtg gatgaggctg gttctaaatc acccattcag tacatcgata tcggtaatta
26041 tacagtttcc tgtttacctt ttacaattaa ttgccaggaa cctaaattgg gtagtcttgt
26101 agtgcgttgt tcgttctatg aagacttttt agagtatcat gacgttcgtg ttgttttaga
26161 tttcatctaa acgaacaaac taaaatgtct gataatggac cccaaaatca gcgaaatgca
26221 ccccgcatta cgtttggtgg accctcagat tcaactggca gtaaccagaa tggagaacgc
26281 agtggggcgc gatcaaaaca acgtcggccc caaggtttac ccaataatac tgcgtcttgg
26341 ttcaccgctc tcactcaaca tggcaaggaa gaccttaaat tccctcgagg acaaggcgtt
26401 ccaattaaca ccaatagcag tccagatgac caaattggct actaccgaag agctaccaga 26461 cgaattcgtg gtggtgacgg taaaatgaaa gatctcagtc caagatggta tttctactac 26521 ctaggaactg ggccagaagc tggacttccc tatggtgcta acaaagacgg catcatatgg 26581 gttgcaactg agggagcctt gaatacacca aaagatcaca ttggcacccg caatcctgct 26641 aacaatgctg caatcgtgct acaacttcct caaggaacaa cattgccaaa aggcttctac 26701 gcagaaggga gcagaggcgg cagtcaagcc tcttctcgtt cctcatcacg tagtcgcaac 26761 agttcaagaa attcaactcc aggcagcagt aggggaactt ctcctgctag aatggctggc 26821 aatggcggtg atgctgctct tgctttgctg ctgcttgaca gattgaacca gcttgagagc 26881 aaaatgtctg gtaaaggcca acaacaacaa ggccaaactg tcactaagaa atctgctgct 26941 gaggcttcta agaagcctcg gcaaaaacgt actgccacta aagcatacaa tgtaacacaa 27001 gctttcggca gacgtggtcc agaacaaacc caaggaaatt ttggggacca ggaactaatc 27061 agacaaggaa ctgattacaa acattggccg caaattgcac aatttgcccc cagcgcttca 27121 gcgttcttcg gaatgtcgcg cattggcatg gaagtcacac cttcgggaac gtggttgacc 27181 tacacaggtg ccatcaaatt ggatgacaaa gatccaaatt tcaaagatca agtcattttg 27241 ctgaataagc atattgacgc atacaaaaca ttcccaccaa cagagcctaa aaaggacaaa 27301 aagaagaagg ctgatgaaac tcaagcctta ccgcagagac agaagaaaca gcaaactgtg 27361 actcttcttc ctgctgcaga tttggatgat ttctccaaac aattgcaaca atccatgagc 27421 agtgctgact caactcaggc ctaaactcat gcagaccaca caaggcagat gggctatata 27481 aacgttttcg cttttccgtt tacgatatat agtctactct tgtgcagaat gaattctcgt 27541 aactacatag cacaagtaga tgtagttaac tttaatctca catagcaatc tttaatcagt 27601 gtgtaacatt agggaggact tgaaagagcc accacatttt caccgaggcc acgcggagta 27661 cgatcgagtg tacagtgaac aatgctaggg agagctgcct atatggaaga gccctaatgt 27721 gtaaaattaa ttttagtagt gctatcccca tgtgatttta atagcttctt aggagaatga 27781 ' (3 3 a 3 cl a a ^^^^3^^3^^ aggccggcat ggtcccagcc tcctcgctgg cgccggctgg 27841 gcaacattcc gaggggaccg tcccctcggt aatggcgaat gggacGGGCC CTGCGATATC 27901 GCGACGAGGA TCTAgatcct ctagagtcga cctgcaggca tgcaagcttg agtattctat 27961 agtctcacct aaatagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 28021 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg 28081 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 28141 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgaacccctt gcggccgccc 28201 gggccgtcga ccaattctca tgtttgacag cttatcatcg aatttctgcc attcatccgc 28261 ttattatcac ttattcaggc gtagcaacca ggcgtttaag ggcaccaata actgccttaa 28321 aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg 28381 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc 28441 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata 28501 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac 28561 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct 28621 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa 28681 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc 28741 agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga 28801 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc 28861 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca 28921 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc 28981 tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac gcccggtagt 29041 gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac gtctcatttt 29101 cgccaaaagt tggcccaggg cttcccggta tcaacaggga caccaggatt tatttattct 29161 gcgaagtgat cttccgtcac aggtatttat tcgcgataag ctcatggagc ggcgtaaccg 29221 tcgcacagga aggacagaga aagcgcggat ctgggaagtg acggacagaa cggtcaggac 29281 ctggattggg gaggcggttg ccgccgctgc tgctgacggt gtgacgttct ctgttccggt 29341 cacaccacat acgttccgcc attcctatgc gatgcacatg ctgtatgccg gtataccgct 29401 gaaagttctg caaagcctga tgggacataa gtccatcagt tcaacggaag tctacacgaa 29461 ggtttttgcg ctggatgtgg ctgcccggca ccgggtgcag tttgcgatgc cggagtctga 29521 tgcggttgcg atgctgaaac aattatcctg agaataaatg ccttggcctt tatatggaaa 29581 tgtggaactg agtggatatg ctgtttttgt ctgttaaaca gagaagctgg ctgttatcca 29641 ctgagaagcg aacgaaacag tcgggaaaat ctcccattat cgtagagatc cgcattatta 29701 atctcaggag cctgtgtagc gtttatagga agtagtgttc tgtcatgatg cctgcaagcg 29761 gtaacgaaaa cgatttgaat atgccttcag gaacaataga aatcttcgtg cggtgttacg 29821 ttgaagtgga gcggattatg tcagcaatgg acagaacaac ctaatgaaca cagaaccatg 29881 atgtggtctg tccttttaca gccagtagtg ctcgccgcag tcgagcgaca gggcgaagcc
29941 ctcgagctgg ttgccctcgc cgctgggctg gcggccgtct atggccctgc aaacgcgcca
30001 gaaacgccgt cgaagccgtg tgcgagacac cgcggccggc cgccggcgtt gtggatacct
30061 cgcggaaaac ttggccctca ctgacagatg aggggcggac gttgacactt gaggggccga
30121 ctcacccggc gcggcgttga cagatgaggg gcaggctcga tttcggccgg cgacgtggag
30181 ctggccagcc tcgcaaatcg gcgaaaacgc ctgattttac gcgagtttcc cacagatgat
30241 gtggacaagc ctggggataa gtgccctgcg gtattgacac ttgaggggcg cgactactga
30301 cagatgaggg gcgcgatcct tgacacttga ggggcagagt gctgacagat gaggggcgca
30361 cctattgaca tttgaggggc tgtccacagg cagaaaatcc agcatttgca agggtttccg
30421 cccgtttttc ggccaccgct aacctgtctt ttaacctgct tttaaaccaa tatttataaa
30481 ccttgttttt aaccagggct gcgccctgtg cgcgtgaccg cgcacgccga aggggggtgc
30541 ccccccttct cgaaccctcc cggtcgagtg agcgaggaag caccagggaa cagcacttat
30601 atattctgct tacacacgat gcctgaaaaa acttcccttg gggttatcca cttatccacg
30661 gggatatttt tataattatt ttttttatag tttttagatc ttctttttta gagcgccttg
30721 taggccttta tccatgctgg ttctagagaa ggtgttgtga caaattgccc tttcagtgtg
30781 acaaatcacc ctcaaatgac agtcctgtct gtgacaaatt gcccttaacc ctgtgacaaa
30841 ttgccctcag aagaagctgt tttttcacaa agttatccct gcttattgac tcttttttat
30901 ttagtgtgac aatctaaaaa cttgtcacac ttcacatgga tctgtcatgg cggaaacagc
30961 ggttatcaat cacaagaaac gtaaaaatag cccgcgaatc gtccagtcaa acgacctcac
31021 tgaggcggca tatagtctct cccgggatca aaaacgtatg ctgtatctgt tcgttgacca
31081 gatcagaaaa tctgatggca ccctacagga acatgacggt atctgcgaga tccatgttgc
31141 taaatatgct gaaatattcg gattgacctc tgcggaagcc agtaaggata tacggcaggc
31201 attgaagagt ttcgcgggga aggaagtggt tttttatcgc cctgaagagg atgccggcga
31261 tgaaaaaggc tatgaatctt ttccttggtt tatcaaacgt gcgcacagtc catccagagg
31321 gctttacagt gtacatatca acccatatct cattcccttc tttatcgggt tacagaaccg
31381 gtttacgcag tttcggctta gtgaaacaaa agaaatcacc aatccgtatg ccatgcgttt
31441 atacgaatcc ctgtgtcagt atcgtaagcc ggatggctca ggcatcgtct ctctgaaaat
31501 cgactggatc atagagcgtt accagctgcc tcaaagttac cagcgtatgc ctgacttccg
31561 ccgccgcttc ctgcaggtct gtgttaatga gatcaacagc agaactccaa tgcgcctctc
31621 atacattgag aaaaagaaag gccgccagac gactcatatc gtattttcct tccgcgatat
31681 cacttccatg acgacaggat agtctgaggg ttatctgtca cagatttgag ggtggttcgt
31741 cacatttgtt ctgacctact gagggtaatt tgtcacagtt ttgctgtttc cttcagcctg
31801 catggatttt ctcatacttt ttgaactgta atttttaagg aagccaaatt tgagggcagt
31861 ttgtcacagt tgatttcctt ctctttccct tcgtcatgtg acctgatatc gggggttagt
31921 tcgtcatcat tgatgagggt tgattatcac agtttattac tctgaattgg ctatccgcgt
31981 gtgtacctct acctggagtt tttcccacgg tggatatttc ttcttgcgct gagcgtaaga
32041 gctatctgac agaacagttc ttctttgctt cctcgccagt tcgctcgcta tgctcggtta
32101 cacggctgcg gcgagcgcta gtgataataa gtgactgagg tatgtgctct tcttatctcc
32161 ttttgtagtg ttgctcttat tttaaacaac tttgcggttt tttgatgact ttgcgatttt
32221 gttgttgctt tgcagtaaat tgcaagattt aataaaaaaa cgcaaagcaa tgattaaagg
32281 atgttcagaa tgaaactcat ggaaacactt aaccagtgca taaacgctgg tcatgaaatg
32341 acgaaggcta tcgccattgc acagtttaat gatgacagcc cggaagcgag gaaaataacc
32401 cggcgctgga gaataggtga agcagcggat ttagttgggg tttcttctca ggctatcaga
32461 gatgccgaga aagcagggcg actaccgcac ccggatatgg aaattcgagg acgggttgag
32521 caacgtgttg gttatacaat tgaacaaatt aatcatatgc gtgatgtgtt tggtacgcga
32581 ttgcgacgtg ctgaagacgt atttccaccg gtgatcgggg ttgctgccca taaaggtggc
32641 gtttacaaaa cctcagtttc tgttcatctt gctcaggatc tggctctgaa ggggctacgt
32701 gttttgctcg tggaaggtaa cgacccccag ggaacagcct caatgtatca cggatgggta
32761 ccagatcttc atattcatgc agaagacact ctcctgcctt tctatcttgg ggaaaaggac
32821 gatgtcactt atgcaataaa gcccacttgc tggccggggc ttgacattat tccttcctgt
32881 ctggctctgc accgtattga aactgagtta atgggcaaat ttgatgaagg taaactgccc
32941 accgatccac acctgatgct ccgactggcc attgaaactg ttgctcatga ctatgatgtc
33001 atagttattg acagcgcgcc taacctgggt atcggcacga ttaatgtcgt atgtgctgct
33061 gatgtgctga ttgttcccac gcctgctgag ttgtttgact acacctccgc actgcagttt
33121 ttcgatatgc ttcgtgatct gctcaagaac gttgatctta aagggttcga gcctgatgta
33181 cgtattttgc ttaccaaata cagcaatagc aatggctctc agtccccgtg gatggaggag
33241 caaattcggg atgcctgggg aagcatggtt ctaaaaaatg ttgtacgtga aacggatgaa 33301 gttggtaaag gtcagatccg gatgagaact gtttttgaac aggccattga tcaacgctct
33361 tcaactggtg cctggagaaa tgctctttct atttgggaac ctgtctgcaa tgaaattttc
33421 gatcgtctga ttaaaccacg ctgggagatt agataatgaa gcgtgcgcct gttattccaa
33481 aacatacgct caatactcaa ccggttgaag atacttcgtt atcgacacca gctgccccga
33541 tggtggattc gttaattgcg cgcgtaggag taatggctcg cggtaatgcc attactttgc
33601 ctgtatgtgg tcgggatgtg aagtttactc ttgaagtgct ccggggtgat agtgttgaga
33661 agacctctcg ggtatggtca ggtaatgaac gtgaccagga gctgcttact gaggacgcac
33721 tggatgatct catcccttct tttctactga ctggtcaaca gacaccggcg ttcggtcgaa
33781 gagtatctgg tgtcatagaa attgccgatg ggagtcgccg tcgtaaagct gctgcactta
33841 ccgaaagtga ttatcgtgtt ctggttggcg agctggatga tgagcagatg gctgcattat
33901 ccagattggg taacgattat cgcccaacaa gtgcttatga acgtggtcag cgttatgcaa
33961 gccgattgca gaatgaattt gctggaaata tttctgcgct ggctgatgcg gaaaatattt
34021 cacgtaagat tattacccgc tgtatcaaca ccgccaaatt gcctaaatca gttgttgctc
34081 ttttttctca ccccggtgaa ctatctgccc ggtcaggtga tgcacttcaa aaagccttta
34141 cagataaaga ggaattactt aagcagcagg catctaacct tcatgagcag aaaaaagctg
34201 gggtgatatt tgaagctgaa gaagttatca ctcttttaac ttctgtgctt aaaacgtcat
34261 ctgcatcaag aactagttta agctcacgac atcagtttgc tcctggagcg acagtattgt
34321 ataagggcga taaaatggtg cttaacctgg acaggtctcg tgttccaact gagtgtatag
34381 agaaaattga ggccattctt aaggaacttg aaaagccagc accctgatgc gaccacgttt
34441 tagtctacgt ttatctgtct ttacttaatg tcctttgtta caggccagaa agcataactg
34501 gcctgaatat tctctctggg cccactgttc cacttgtatc gtcggtctga taatcagact
34561 gggaccacgg tcccactcgt atcgtcggtc tgattattag tctgggacca cggtcccact
34621 cgtatcgtcg gtctgattat tagtctggga ccacggtccc actcgtatcg tcggtctgat
34681 aatcagactg ggaccacggt cccactcgta tcgtcggtct gattattagt ctgggaccat
34741 ggtcccactc gtatcgtcgg tctgattatt agtctgggac cacggtccca ctcgtatcgt
34801 cggtctgatt attagtctgg aaccacggtc ccactcgtat cgtcggtctg attattagtc
34861 tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acgatcccac
34921 tcgtgttgtc ggtctgatta tcggtctggg accacggtcc cacttgtatt gtcgatcaga
34981 ctatcagcgt gagactacga ttccatcaat gcctgtcaag ggcaagtatt gacatgtcgt
35041 cgtaacctgt agaacggagt aacctcggtg tgcggttgta tgcctgctgt ggattgctgc
35101 tgtgtcctgc ttatccacaa cattttgcgc acggttatgt ggacaaaata cctggttacc
35161 caggccgtgc cggcacgtta accgggctgc atccgatgca agtgtgtcgc tgtcgacgag
35221 ctcgcgagct cggacatgag gttgccccgt attcagtgtc gctgatttgt attgtctgaa
35281 gttgttttta cgttaagttg atgcagatca attaatacga tacctgcgtc ataattgatt
35341 atttgacgtg gtttgatggc ctccacgcac gttgtgatat gtagatgata atcattatca
35401 ctttacgggt cctttccggt gatccgacag gttacggggc ggcgacctcg cgggttttcg
35461 ctatttatga aaattttccg gtttaaggcg tttccgttct tcttcgtcat aacttaatgt
35521 ttttatttaa aataccctct gaaaagaaag gaaacgacag gtgctgaaag cgagcttttt
35581 ggcctctgtc gtttcctttc tctgtttttg tccgtggaat gaacaatgga agtccgagct
35641 catcgctaat aacttcgtat agcatacatt atacgaagtt atattcgat
//
As used herein, the terms “RNA replicon” or “replicon RNA” refer to RNA, which contains all of the genetic information required for directing its own amplification or self-replication within a permissive cell. To direct its own replication, the RNA molecule (1) encodes polymerase, replicase, or other proteins which may interact with viral or host cell-derived proteins, nucleic acids or ribonucleoproteins to catalyze the RNA amplification process; and (2) contain cis-acting
RNA sequences required for replication and transcription of the subgenomic replicon-encoded RNA. These sequences may be bound during the process of replication to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids or ribonucleoproteins, or complexes between any of these components. A coronavims-derived replicon RNA molecule typically contains the following ordered elements: 5’ viral or defective-interfering RNA sequence(s) required in cis for replication, sequences coding for biologically active coronavirus nonstructural proteins (e.g., nsPl, nsP2, nsP3, and nsP4), promoter for the subgenomic RNA, 3’ viral sequences required in cis for replication, and a polyadenylate tract. Further, the term RNA replicon generally refers to a molecule of positive polarity or “message” sense, and the RNA replicon may be of length different from that of any known, naturally-occurring coronavirus.
In some embodiments, the RNA replicon does not contain the sequences of at least a spike protein. For example, the coding sequence of the spike protein can be substituted with heterologous sequences. In some embodiments, the RNA replicon may be packaged into a recombinant coronavirus particle, and it may include one or more sequences, such as packaging signals, which serve to initiate interactions with coronavirus structural proteins that lead to particle formation.
As used herein, “subgenomic RNA” refers to an RNA molecule of a length or size which is smaller than the genomic RNA from which it was derived. The coronavirus subgenomic RNA should be transcribed from an internal promoter, whose sequences reside within the genomic RNA or its complement. Transcription of a coronavirus subgenomic RNA may be mediated by the viral- encoded polymerase(s) associated with host cell-encoded proteins, ribonucleoprotein(s), or a combination thereof. In some embodiments, the subgenomic RNA is produced from a modified RNA replicon, as disclosed herein.
In some embodiments, a part or the entire coding sequence for the spike protein is absent and/or modified in the nucleic acid molecules disclosed herein. In some embodiments, the modified coronavirus genome or RNA replicon can be devoid of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the sequence encoding the spike protein. In some embodiments, the modified coronavirus genome or RNA replicon is devoid of a substantial portion of or the entire sequence encoding the spike protein. As used herein, a “substantial portion” of a nucleic acid sequence comprises enough of the nucleic acid sequence encoding the viral structural protein to afford putative identification of that protein, either by manual evaluation of the sequence by one skilled in the art or by computer-automated sequence comparison and identification using algorithms such as BLAST (see, for example, Altschul SF el al. 1993, supra). In some embodiments, the modified coronavirus genome or RNA replicon is devoid of the entire sequence encoding the spike protein.
In addition, the nucleic acid molecules may include a modified coronavirus genome or RNA replicon containing one or more attenuating mutations so as to increase the safety of virus manipulation and/or administration. The term “attenuating mutation” as used herein means a nucleotide mutation or an amino acid encoded in view of such mutation, which results in a decreased probability of causing disease in its host (i.e., a loss of virulence), in accordance with standard terminology in the art, whether the mutation is a substitution mutation or an in-frame deletion or insertion mutation. Attenuating mutations may be in the coding or non-coding regions of the coronavirus genome. As known by those skilled in the art, the term “attenuating mutation” excludes mutations or combinations of mutations that would be lethal to the virus. Further, those skilled in the art will appreciate that some attenuating mutations may be lethal in the absence of a second-site suppressor mutation(s).
In some embodiments, the nucleic acid molecules are recombinant nucleic acid molecules. As used herein, the term “recombinant” means any molecule (e.g., DNA, RNA, polypeptide) that results from human manipulation. As an example, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector. For example, a recombinant nucleic acid molecule: (1) can be synthesized or modified in vitro , for example, using chemical or enzymatic techniques (e.g., by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, exonucleolytic digestion, endonucleolytic digestion, ligation, reverse transcription, transcription, base modification (including, e.g., methylation), or recombination (including homologous and site- specific recombination) of nucleic acid molecules; (2) may include conjoined nucleotide sequences that are not conjoined in nature; (3) can be engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleotide sequence; and/or (4) may be manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleotide sequence.
In some embodiments, the nucleic acid molecules disclosed herein are produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. The nucleic acid molecules may include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and/or substituted in such a manner that such modifications provide the desired property in effecting a biological activity as described herein.
A nucleic acid molecule, including a variant of a naturally-occurring nucleic acid sequence, can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et ah, In: Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). The sequence of a nucleic acid molecule can be modified with respect to a naturally-occurring sequence from which it is derived using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as but not limited to site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, recombinational cloning, and chemical synthesis, including chemical synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologs can be selected from a mixture of modified nucleic acid molecules by screening for the function of the protein or the replicon encoded by the nucleic acid molecule and/or by hybridization with a wild-type gene or fragment thereof, or by PCR using primers having homology to a target or wild-type nucleic acid molecule or sequence.
In some embodiments, the modified coronavirus genome or RNA replicon is operably linked to a heterologous regulatory element. As used herein, “regulatory element,” “regulatory sequence,” or “regulatory element sequence” refers to a nucleotide sequence located upstream (5’), within, or downstream (3’) of a coding sequence such as, for example, a polypeptide-encoding sequence or a functional RNA-encoding sequence. Transcription of the coding sequence and/or translation of an RNA molecule resulting from transcription of the coding sequence is typically affected by the presence or absence of the regulatory element. These regulatory elements may comprise promoters, cis-elements, enhancers, terminators, or introns. One of skill in the art will appreciate that the regulatory elements described herein may be present in a chimeric or hybrid regulatory expression element. In some embodiments, the heterologous regulatory element is, or comprises, a promoter sequence. The heterologous promoter sequence can be any heterologous promoter sequence, for example, a SP6 promoter, a T3 promoter, or a T7 promoter, or a combination thereof. In some embodiments, the promoter sequence includes a T7 promoter sequence.
Further, in some embodiments, the modified coronavirus genome or RNA replicon can include one or more heterologous transcriptional termination signal sequences. The term “transcriptional termination signal,” “terminator” or “terminator sequence” or “transcription terminator,” as used interchangeably herein, refers to a regulatory section of genetic sequence that causes RNA polymerase to cease transcription. The heterologous transcriptional termination signal sequences can generally be any heterologous transcriptional termination signal sequences, and for example, a SP6 termination signal sequence, a T3 termination signal sequence, a T7 termination signal sequence, or a variant thereof. Accordingly, In some embodiments, the nucleic acid molecules can include at least one of the one or more heterologous transcriptional termination signal sequences selected from the group consisting of a SP6 termination signal sequence, a T3 termination signal sequence, a T7 termination signal sequence, or a variant thereof. In some particular embodiments, the transcriptional termination sequence includes a T7 termination signal sequence.
In some embodiments, the nucleic acid molecules disclosed herein can include one or more expression cassettes. In some particular embodiments, the nucleic acid molecules can include at least two, at least three, at least four, at least five, or at least six expression cassettes. As used herein, the term “expression cassette” refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo. The expression cassette may be inserted into a vector for targeting a desired host cell and/or into a subject. Further, the term expression cassette may be used interchangeably with the term “expression construct.” In some embodiments, the term “expression cassette” refers to a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as, for example, a promoter and/or a termination signal, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the gene. The term “operably linked,” as used herein, denotes a functional linkage between two or more sequences. For example, an operably linkage between a polynucleotide of interest and a regulatory sequence (for example, a promoter) is a functional link that allows for expression of the polynucleotide of interest. In this sense, the term “operably linked” refers to the positioning of a regulatory region and a coding sequence to be transcribed so that the regulatory region is effective for regulating transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term “operably linked” denotes a configuration in which a regulatory sequence is placed at an appropriate position relative to a sequence that encodes a polypeptide or functional RNA such that the control sequence directs or regulates the expression or cellular localization of the mRNA encoding the polypeptide, the polypeptide, and/or the functional RNA. Thus, a promoter is in operable linkage with a nucleic acid sequence if it can mediate transcription of the nucleic acid sequence. Operably linked elements may be contiguous or non-contiguous. The techniques for operably linking two or more sequences of DNA together are familiar to the skilled worker, and such methods have been described in a number of texts for standard molecular biological manipulation (see, for example, Maniatis et ah, “Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Gibson et al, Nature Methods 6:343-45, 2009).
In some embodiments, the disclosed nucleic acid molecules may include a codon- optimized sequence. For example, the nucleic acid sequence may be codon-optimized for expression in a eukaryote or eukaryotic cell. In some embodiments, the codon-optimized nucleic acid sequence is codon-optimized for operability in a eukaryotic cell or organism, e.g. , a yeast cell, or a mammalian cell or organism, including a mouse cell, a rat cell, and a human cell or non-human eukaryote organism.
Generally, codon optimization refers to a process of modifying a nucleic acid sequence to enhance expression in the host cells by substituting at least one codon of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit a particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., el al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa ). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, ormore, or all codons) in a sequence encoding a DNA/RNA-targeting IL-2 variant corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at http://www.yeastgenome.org/community/codonusage.shtml, or Codon selection in yeast , Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31. As to codon usage in plants including algae, reference is made to Codon usage in higher plants, green algae, and cyanobacteria , Campbell and Gowri, Plant Physiol. 1990 January; 92(1): 1-11.; as well as Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan. 25; 17(2):477-98; or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton B R, J Mol Evol. 1998 April; 46(4):449-59.
“Coronavirus ,” as used herein, refers to a genus in the family Coronaviridae, which family is in turn classified within the order Nidovirales. The coronaviruses are large, enveloped, positive- stranded RNA viruses. They have the largest genomes of all RNA viruses and replicate by a unique mechanism that results in a high frequency of recombination. The coronaviruses include antigenic groups I, II, and III. Nonlimiting examples of coronaviruses include SARS coronavirus ( i.e ., SARS-CoV, SARS-CoV-2), MERS coronavirus, transmissible gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, murine hepatitis virus, sialodacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as chimeras of any of the foregoing. See Lai and Holmes “ Coronaviridae: The Viruses and Their Replication” in Fields Virology, (4th Ed. 2001). In some embodiments, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
In another aspect, this disclosure also provides a virus particle or a virus-like particle comprising a nucleic acid molecule described above. In some embodiments, the virus particle or virus-like particle comprises a vesicular stomatitis virus G (VSV-G) protein.
As used herein, the terms “sub-viral particle,” “virus-like particle,” “recombinant subviral particles” or “VLP” refer to a nonreplicating, viral shell. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art and discussed more fully below. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical and immunological characterizations, and the like. See , e.g., Baker el al. , Biophys. J. (1991) 60:1445-1456; Hagensee et al, J. Virol. (1994) 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation and images recorded under appropriate exposure conditions. Additional methods of VLP purification include but are not limited to chromatographic techniques such as affinity, ion exchange, size exclusion, and reverse-phase procedures.
B. CELLS AND COMPOSITIONS
In another aspect, this disclosure further provides a cell or cell line comprising a nucleic acid molecule described above. In some embodiments, the cell or cell line further comprises a second nucleic acid molecule comprising a coding sequence of a VSV-G protein or a variant/fragment thereof.
In some embodiments, the VSV-G protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or comprises the amino acid sequence of SEQ ID NO: 2.
In some embodiments, the cell or cell line further comprises a third nucleic acid molecule comprising a coding sequence of a Spike protein or a variant/fragment thereof. In some embodiments, the Spike protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or comprises the amino acid sequence of SEQ ID NO: 4.
A representative amino acid sequence of the Spike protein is provided below (Accession ID: NC_045512.2; SEQ ID NO: 4):
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNA TNW IKVCEFQFCNDPFLGVYYHKNNKSMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFK NLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTP GDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQT SNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKC YGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQP YRW VLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIAD TTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRV YSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSL GAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQL NRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVT LADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAG AALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDW NQN AQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIR ASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGW FLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDW IGIVNNTVYDPLQPELDS FKEELDKYFKNHTSPDVDLGDISGINASW NIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK WP YIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
Also provided in this disclosure is a method of preparing a coronavirus replicon-harbored cell. The method may include introducing a nucleic acid molecule described above to a host cell, such as an animal cell. In some embodiments, the method may further include culturing the cell in a cell culture medium to obtain the coronavirus replicon-harbored cell. In some embodiments, the method further comprises introducing to the cell a second nucleic acid comprising a coding sequence of the YSV-G protein or a variant/fragment thereof. Alternationally and/or additionally, the method may further comprise introducing to the cell a third nucleic acid comprising a coding sequence of the Spike protein or a variant/fragment thereof.
The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. In some embodiments, the nucleic acid molecule is introduced into a host cell by an electroporation procedure or a biolistic procedure.
In some embodiments, host cells can be genetically engineered (e.g, transduced or transformed or transfected) with, for example, a vector construct of this disclosure. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. The vector containing a polynucleotide sequence as described herein, e.g, a nucleic acid molecule comprising a modified coronavirus genome or RNA replicon, as well as, optionally, a selectable marker or reporter gene, can be employed to transform an appropriate host cell.
The methods disclosed herein may be deployed for genetic engineering of any species, including, but not limited to, prokaryotic and eukaryotic species. Suitable host cells can include, but are not limited to, algal cells, bacterial cells, heterokonts, fungal cells, chytrid cells, microfungi, microalgae, and animal cells. In some embodiments, the animal cells are invertebrate animal cells. In some embodiments, the vertebrate animal cells are mammalians cells. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.
In some embodiments, the methods disclosed herein can be used with host cells from species that are natural hosts of coronaviruses, such as rodents, mice, fish, birds, and larger mammals such as humans, horses, pig, monkey, and apes, as well as invertebrates. In principle, any animal species can be generally used and can be, for example, human, dog, bird, fish, horse, pig, primate, mouse, cattle, swine, sheep, rabbit, cat, goat, donkey, hamster, or buffalo. Non limiting examples of suitable bird species include chicken, duck, goose, turkey, ostrich, emu, swan, peafowl, pheasant, partridge, and guinea fowl. In some particular embodiments, the fish species is a salmon species. Primary mammalian cells and continuous/immortalized cells types are also suitable. Non-limiting examples of suitable animal host cells include, but not limited to, pulmonary equine artery endothelial cell, equine dermis cell, baby hamster kidney (BHK) cell, rabbit kidney cell, mouse muscle cell, mouse connective tissue cell, human cervix cell, human epidermoid larynx cell, Chinese hamster ovary cell (CHO), human HEK-293 cell, mouse 3T3 cell, Vero cell, Madin- Darby Canine Kidney Epithelial Cell (MDCK), primary chicken fibroblast cell, a HuT78 cell, a Huh-7 cell, A549 lung cell, HeLa cell, PER.C6® cell, WI-38 cell, MRC-5 cell, FRhL-2, and CEM T-cell. In some embodiments, the host cell is a baby hamster kidney cell. In some embodiments, the baby hamster kidney cell is a BHK-21 cell. In some embodiments, the host cell is a Huh-7 cell or derived from a Huh-7 cell. In some embodiments, the host cell is a Huh-7.5 cell. In some embodiments, the cell is a lung organoid.
Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures, including at least one host cell disclosed herein, are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.
Also within the scope of this disclosure is a composition comprising a nucleic acid molecule or a cell or cell line, as described above, and a pharmaceutically acceptable carrier.
As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
The term “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subj ect such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.
In another aspect, this disclosure provides a kit comprising a nucleic acid molecule, a cell or cell line, or the composition, as described above. In addition, the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The composition can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile. When the components of the kit are provided in a liquid solution, the liquid solution preferably is an aqueous solution. When the agents are provided as a dried form, reconstitution generally is by the addition of a suitable solvent and acidulant. The acidulant and solvent, e.g., an aprotic solvent, sterile water, or a buffer, can optionally be provided in the kit. In some embodiments, the kit may further include informational materials. The informational material of the kits is not limited in its form. For example, the informational material can include information about the production of the composition, concentration, date of expiration, batch or production site information, and so forth.
C. METHODS OF USE
The above-described RNA replicons can be used as a low-containment platform for molecular virology studies and drug development screening. Accordingly, this disclosure further provides a method for screening for antiviral agents for a coronavirus.
In some embodiments, the method comprises: (i) contacting a cell or cell line described above with a candidate agent (e.g., test compound); and (ii) determining an increase or decrease in replication or activity of the coronavirus virus replicon relative to a control cell or cell line harboring the same replicon, wherein the control cell or cell line has not been contacted with the candidate agent. In some embodiments, the coronavirus is SARS-CoV or SARS-CoV-2. In some embodiments, the step of determining comprises determining a level of production of a coronavirus protein or a coronavirus RNA transcript.
In some embodiments, antiviral agents can be an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate. Examples of such a test compound include small organic or inorganic molecules, proteins, peptides, peptidomimetics, polysaccharides, nucleic acids, nucleic acid analogues and derivatives, or peptoids. Candidate compounds to be screened (e.g., proteins, peptides, peptidomimetics, peptoids, antibodies, small molecules, or other drugs) can be isolated from naturally occurring substances or obtained using any of the numerous approaches in combinatorial library methods known in the art. Such libraries include: peptide libraries, peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that is resistant to enzymatic degradation); spatially addressable parallel solid phase or solution phase libraries; synthetic libraries obtained by deconvolution or affinity chromatography selection; and the “one-bead one-compound” libraries. See, e.g., Zuckermann et al. 1994, J. Med. Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145. Examples of methods for the synthesis of molecular libraries can be found in, e.g, DeWitt etal, 1993, PNAS USA 90:6909; Erb etal, 1994, PNAS USA 91:11422; Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al, 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell etal, 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al, 1994 J. Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (U.S. Patent No. 5,223,409), plasmids (Cull etal, 1992, PNAS USA 89:1865- 1869), or phages (Scott and Smith 1990, Science 249:386-390; Devlin, 1990, Science 249:404- 406; Cwirla et al, 1990, PNAS USA 87:6378-6382; Felici 1991, J. Mol. Biol. 222:301-310; and U.S. Patent No. 5,223,409).
A candidate compound/composition identified by the evaluation method can be further tested to confirm its therapeutic effect or modified to optimize its effect and limit any side effects, and then formulated as a therapeutic agent. Therapeutic agents thus identified can be used in a therapeutic protocol to treat coronavirus infection. The term “determining” means methods that include detecting the presence or absence or a level of marker(s) ( e.g ., a coronavirus protein or a coronavirus RNA transcript) in the sample, quantifying the amount of marker(s) in the sample, and/or qualifying the type of biomarker. Measuring can be accomplished by methods known in the art and those further described herein.
The level of the one or more markers in a sample obtained from a subject may be determined by any of a wide variety of well-known techniques and methods, which transform a marker within the sample into a moiety that can be detected and quantified. Non-limiting examples of such methods include analyzing the sample using immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods, immunoblotting, Western blotting, Northern blotting, electron microscopy, mass spectrometry, e.g., MALDI-TOF and SELDI-TOF, immunoprecipitations, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assays (ELISAs), e.g., amplified ELISA, quantitative blood-based assays, e.g., serum ELISA, quantitative urine-based assays, flow cytometry, Southern hybridizations, array analysis, and the like, and combinations or sub combinations thereof.
In some embodiments, the level of a marker in a sample can be determined by detecting a transcribed polynucleotide or portion thereof, e.g., mRNA, or cDNA, of a marker gene. RNA may be extracted from cells using RNA extraction techniques, including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton el al, (1984) Nuc. Acids Res. 12:7035-56), Northern blotting, in situ hybridization, and microarray analysis.
More than one antiviral agents, e.g. , a plurality of test compounds, can be tested at the same time for their ability to modulate the expression and/or activity of a marker in a screening assay. The term “screening assay” refers to assays that test the ability of a plurality of compounds to influence the readout of choice rather than to tests that test the ability of one compound to influence a readout. In some embodiments, the assays may identify compounds not previously known to have the effect that is being screened for. In some embodiments, high throughput screening (HTS) can be used to assay for the activity of a compound.
D. DEFINITIONS
To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
The term “gene” is used broadly to refer to any segment of a nucleic acid molecule that encodes a protein or that can be transcribed into a functional RNA. Genes may include sequences that are transcribed but are not part of a final, mature, and/or functional RNA transcript, and genes that encode proteins may further comprise sequences that are transcribed but not translated, for example, 5’ untranslated regions, 3’ untranslated regions, introns, etc. Further, genes may optionally further comprise regulatory sequences required for their expression, and such sequences may be, for example, sequences that are not transcribed or translated. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.
The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules can have any three-dimensional structure. A nucleic acid molecule can be double- stranded or single-stranded (e.g., a sense strand or an antisense strand). Non-limiting examples of nucleic acid molecules include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, micro-RNA, tracrRNAs, crRNAs, guide RNAs, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, nucleic acid probes, and nucleic acid primers. A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein interchangeably refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases as set forth in 37 CFR § 1.822 is used herein.
Nucleic acid molecules can be nucleic acid molecules of any length, including but not limited to, nucleic acid molecules that are between about 3 Kb and about 50 Kb, for example, between about 3 Kb and about 40 Kb, between about 3 Kb and about 40 Kb, between about 3 Kb and about 30 Kb, between about 3 Kb and about 20 Kb, between 5 Kb and about 40 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb. The nucleic acid molecules can also be, for example, more than 50 kb.
The polynucleotides of the present disclosure can be “biologically active” with respect to either a stmctural attribute, such as the capacity of a nucleic acid to hybridize to another nucleic acid, or the ability of a polynucleotide sequence to be recognized and bound by a transcription factor and/or a nucleic acid polymerase.
Typical “control elements” include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), and translation termination sequences, and/or sequence elements controlling an open chromatin structure see e.g., McCaughan etal. (1995) PNAS USA 92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355.
As used herein, the term “construct” is intended to mean any recombinant nucleic acid molecule such as an expression cassette, plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single- stranded or double- stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g. operably linked.
As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene products.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
The terms “cells,” “cell cultures,” “cell line,” “recombinant host cells,” “recipient cells” and “host cells,” as used herein, include the primary subject cells and any progeny thereof, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell (due to deliberate or inadvertent mutations or differences in environment); however, such altered progeny are included in these terms, so long as the progeny retain the same functionality as that of the originally transformed cell.
As used herein, the term “variant” refers to a first molecule that is related to a second molecule (also termed a “parent” molecule). The variant molecule can be derived from, isolated from, based on or homologous to the parent molecule. A “functional variant” of a protein as used herein refers to a variant of such protein that retains at least partially the activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or replacement mutants), including polymorphs, etc. Also included within functional variants are fusion products of such protein with another, usually unrelated, nucleic acid, protein, polypeptide, or peptide. Functional variants may be naturally occurring or may be man-made. As used herein, the term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); beta-branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) includes one or more conservative modifications. The Cas protein with one or more conservative modifications may retain the desired functional properties, which can be tested using the functional assays known in the art.
As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The term “homolog” or “homologous,” when used in reference to a polypeptide, refers to a high degree of sequence identity between two polypeptides, or to a high degree of similarity between the three-dimensional structure or to a high degree of similarity between the active site and the mechanism of action. In some embodiments, a homolog has a greater than 60% sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence. The term “substantial identity,” as applied to polypeptides, means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 75% sequence identity.
A peptide or polypeptide “fragment” as used herein refers to a less than full-length peptide, polypeptide or protein. For example, a peptide or polypeptide fragment can have at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids in length, or single unit lengths thereof. For example, fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length. There is no upper limit to the size of a peptide fragment. However, in some embodiments, peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less than about 250 amino acids in length.
In some embodiments, variants and homologs may have sequences with at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the sequences of transgenes described herein.
The term “disease” as used herein is intended to be generally synonymous and is used interchangeably with, the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.
As used herein, the term “modulate” is meant to refer to any change in biological state, i.e., increasing, decreasing, and the like. The terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced,” “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example, a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
The terms “increased,” “increase” or “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,” “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
The terms “therapeutic agent,” “therapeutic capable agent,” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition. “Sample,” “test sample,” and “patient sample” may be used interchangeably herein. The sample can be a sample of, serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells (e.g., antibody-producing cells) or tissue. Such a sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art. The terms “sample” and “biological sample” as used herein generally refer to a biological material being tested for and/or suspected of containing an analyte of interest such as antibodies. The sample may be any tissue sample from the subject. The sample may comprise protein from the subject.
The terms “inhibit” and “antagonize,” as used herein, mean to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein’s expression, stability, function or activity by a measurable amount or to prevent entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down-regulate a protein, a gene, and mRNA stability, expression, function and activity, e.g., antagonists.
As used herein, the term “in vitro' ’ refers to events that occur in an artificial environment, e.g, in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
As used herein, the term “in vivo ” refers to events that occur within a multi-cellular organism, such as a non-human animal.
It is noted here that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
The terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.
The phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise. The terms “and/or”
Figure imgf000055_0001
means any one of the items, any combination of the items, or all of the items with which this term is associated.
The word “substantially” does not exclude “completely,” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.
As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.
It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.
As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise. In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.
Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. E. EXAMPLES
EXAMPLE 1
This example describes the materials and methods used in EXAMPLE 2. Replicon fragment cloning
Table 1 contains the primers used for fragment cloning.
Table 1. List of primers for SARS-CoV-2 replicon construction - Organized by PCR reaction, final fragments for subcloning and yeast transformation are highlighted in bold, all others are intermediate overlap PCR templates.
Figure imgf000057_0001
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DNA fragments 2-6 and 8 were the same as described in Thao et al. (PMID: 32365353). The rest of the fragments were PCR-amplified from SARS-CoV-2 clone 3.1 (Thao et al. PMID: 32365353), except fragment 7, which was amplified from cDNA obtained by RT-PCR of viral RNA extracted from isolate USA-WA1/2020 (BEI #NR-5281), grown in Vero-E6 cells. RNA was extracted from cells using Trizol (ThermoFisher #15596026) and RNeasy mini kit (Qiagen #74104), and cDNA was prepared using Superscript™ IV First-Strand Synthesis System with random primers (Thermo #18091050).
PCR amplification reactions were performed using KOD Xtreme Hot Start DNA Polymerase (EMD Millipore #71975). Accessory sequences, such as Neon Green, Glue, NeoR, were amplified from plasmids or purchased as synthetic DNA (IDT). PCR amplicons of fragments
1, 9, 10, 11, 12, and 13 were phosphorylated with T4 PNK (NEB #M0201) and ligated directly into pGEM3Z plasmid, made linear via PCR (T4 DNA ligase, NEB #M0202T). Fragment 7 pol(- ) was similarly cloned into pACNR-2015FLYF\17D (PMID: 32980446), a low-copy plasmid. SARS-CoV-2 N protein was amplified by PCR to include polyA sequence at the 3’ end and was cloned into Kpnl+Xbal digested PGEM3Z plasmid using Gibson assembly.
Fragments-containing pUC, pUC mini, and PGEM3Z plasmids were grown in E. coli DH5 a (ThermoFisher), and pACNRand pCCl-BAC-His3 were grown in E.coh MC I 061 Plasmid DNA was extracted with ZymoPURE II Plasmid Maxiprep (Zymo research #D4202).
Mutagenesis of SARS-CoV-2 nspl and nspl2 was done using the two-step PCR method on fragments 2 and 7, respectively, using the primers listed in Table 1. Pol(-) mutant was created by mutating SARS-CoV-2 RdRp (nspl2) D760, D761 catalytic residues to N760, N761 (SDD/SNN) (41). Nspl mutant that does not bind the 40S ribosome was created by mutating K164A/H165A (28, 29). The mutated fragments were used for replicon assembly, as detailed below. Yeast assembly and bacterial propagation of assembled replicons
DNA fragments for assembly were prepared by restriction digestion or PCR as detailed in Table 2, and agarose gel was extracted. Table 2 Preparation of fragments for yeast transformation associated recombination
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Yeast assembly was performed according to the protocol in Thao eial., 2020 (42). Briefly, 50-100 ng of each fragment was mixed in an equimolar ratio and transformed into Saccharomyces cerevisiae (S. cerevisiae) strain VL6-48N. Transformed yeast was grown for 2-3 days on selective -HIS plates at 30°C. 4-10 colonies from each plate were picked, re-streaked on a new plate, and grown for 2 days at 30°C. Crude DNA extraction was performed using glass beads (Biospec #11079110) and Chelex® (SigmaAldrich #95577-100G-F) and screened by multiplex PCR with the primers detailed in Table 1, using multiplex PCR kit (Qiagen #206143) according to the manufacturer’s protocol. Fragment 7, which contains the coding sequence for the viral RdRp, was amplified by PCR from the positive clones and sequence-verified by sanger sequencing.
For large-scale plasmid extraction from yeast, the protocol in Thao et al, 2020 was used as described, except ZymoPURE II Plasmid Maxiprep (Zymo research #D4202) instead.
To eliminate yeast chromosomal DNA, the plasmid prep was digested with BamHI-HF enzyme (NEB #R3136T), which does not have restriction sites in any of the pCCl-BAC-His3- replicon plasmids. Next, DNA was digested with Plasmid-Safe™ ATP -Dependent DNase (Lucigen #E3101K) for 24h and cleaned by extraction with Phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation. Final DNA concentration was measured using Qbit dsDNA HS Assay (ThermoFisher #Q32851)
For bacterial propagation, 1 mΐ of the crude yeast DNA prep was electroporated into E. coli TransforMax™ Epi300™ electrocompetent cells (Lucigen #EC300110). The bacteria were then plated on chloramphenicol-containing plates and grown at 30°C overnight. Bacterial clones were used to make 5ml starters cultures and grown overnight at 30°C. The following day, the starter was diluted 1:10 in fresh LB+Choramphenicol with copy-control solution (Lucigen #CCIS125) and incubated in a shaker incubator at 37C for 5h. Plasmids were then extracted using a Qiaprep spin miniprep kit (Qiagen #27104). As with the yeast preps, fragment 7 was amplified by PCR sequence-verified by sanger sequencing.
Multiple displacement amplification (MPA) of replicon plasmids and DNA template preparation. For amplification of pCCl-BAC-His3-replicon plasmids, 5-30 ng of DNA was used with EquiPhi29™ DNA Polymerase (ThermoFisher #A39390) according to manufacturer’s instructions, using exonuclease-resistant random primers (ThermoFisher #S0181).
Amplified DNA was digested with Notl-HF enzyme (NEB #R3189S) and cleaned up with phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation.
In vitro transcription and electroporation ofRNA
Transcription reactions were set up using 1 pg linear DNA templates using a HiScribe™ T7 High Yield RNA Synthesis Kit (NEB #E2040S) or T7 RiboMAX™ Express Large Scale RNA Production System (Promega #P1320). After in-vitro transcription, RNA was treated with Ambion™ DNase I (ThermoFisher #AM2222) and cleaned using Monarch® RNA Cleanup Kit (NEB #T2050L). For co-transcriptional capping ofRNA, Anti-Reverse Cap Analog (ARCA) was used at 1:2.8 (GTP: ARCA) ratio (NEB #S 1411 S). For post-transcriptional capping, CellScript ScriptCap™ Cap 1 Capping System was used with ScriptCap™ 2’-0-Methyltransferase to generate Capl, or without ScriptCap™ 2’-0-Methyltransferase to generate CapO. Concentration and quality were measured by nanodrop. To visualize the size of the RNA product, RNA was loaded on 0.8% agarose gel and Gel Loading Buffer II (ThermoFisher #AM8546G).
Sindbis replicon RNA was in-vitro transcribed from a SinRep5-GFP plasmid linearized with Xhol (NEB) (6), using SP6 mMessage mMachine High Yield Capped RNA Transcription kit (ThermoFisher #AM1340) RNA transcripts were electroporated into Huh7.5 or BHK-21 cells using adapted protocols originally developed for launching HCV (43). Briefly, Huh7.5 or BHK-21 cells were trypsinized, washed twice with ice-cold phosphate-buffered saline (PBS) (Invitrogen), and resuspended at 1.5 x 107 cells/ml in PBS. Subsequently, 5 pg of SARS-CoV-2 replicon RNA and 2 pg of SARS- CoV2 N mRNA were mixed with 0.4 ml of cell suspension in a 2-mm cuvette (BTX #45-0125) and immediately pulsed using a BTX ElectroSquare Porator ECM 830 (860V, 99 ps, five pulses). Electroporated cells were incubated at room temperature for 10 min prior to resuspension in plating media.
Cell culture Huh-7.5 cells (H. sapiens ; sex: male) (44), Caco-2 (ATCC® HTB-37™, H. sapiens, sex: male), A549 (ATCC® CCL-185™, H. sapiens, sex: male), and VeroE6 cells (ATCC® CRL- 1586™, C. aethiops) were maintained at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, Fisher Scientific, cat. #11995065) supplemented with 0.1 mM nonessential amino acids (NEAA, Fisher Scientific, cat. #11140076) and 10% hyclone fetal bovine serum (FBS, HyClone Laboratories, Lot. #AUJ35777). BHK-21 cells (ATCC CCL-10, M. auratus ) were grown in Minimum Essential Medium (MEM, Fisher Scientific, cat. #11095080), supplemented with 8% FBS and 0.1 mMNEAA. Calu-3 cells (ATCC® HTB-55™, H. sapiens ; sex: male) were grown in Eagle’s Minimum Essential Medium (EMEM, ATCC® 30-2003™) supplemented with 10% FBS and 0.1 mM NEAA on collagen-coated dishes. Normal Human Lung Fibroblasts (NHLF, Lonza #CC-2512) and Normal Human Bronchial Epithelial cells (NHBE, Lonza #CC-2541) were subcultured using Reagent Pack™ Subculture Reagents (Lonza #CC-5034) and maintained in FGM™-2 Fibroblast Growth Medium-2 BulletKit™ (Lonza #CC-3132) and BEGM™-2 BulletK.it™ Medium (Lonza #CC-3170) respectively. TMEM41B-KO and dox-inducible TMEM4 IB-reconstituted Huh-7.5 cells were previously described (45). TMEM41B expression was induced by Doxy cy cline at least 24h before electroporation.
All cell lines have tested negative for contamination with mycoplasma.
Secreted gaussia luciferase assays Following electroporation, between 15-30K cells in 100 pi media per well were plated into
96 well plates containing equal volumes (IOOmI) of indicated compound dilutions at 2x concentrations. Cell supernatants were cumulatively harvested at various timepoints or at 24hrs post-electroporation, and luciferase signal was measured using the Renilla luciferase assay system (Promega, E2820) in a Fluostar Omega microplate reader (BMG Labtech). Cell viability was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega #G7570) following the manufacturer’s instructions. aPCR
Huh7.5 cells containing repSARS-CoV-2 Glue, minirepSARS-CoV-2 Glue, or repSARS- CoV-2 Glue pol- replicons were seeded onto 12-well plates in triplicate at 1 x 105 cells/well and treated with lOOnM remdesivir or DMSO vehicle. After incubating for 24 or 48 hours at 37 °C, supernatants were aspirated, cells were washed three times with PBS and subsequently lysed in 250 mΐ Tri-reagent (Zymo, cat. #R2050) per well. RNA was extracted using the Direct-zol RNA Miniprep Plus kit (Zymo Research, cat. #R2072) according to the manufacturer’s protocol, followed by reverse transcription into cDNA using random hexamer primers with the Superscript III First-Strand Synthesis System Kit (Invitrogen, cat. #18080051) following the manufacturer’s instructions. Gene expression was quantified by qRT-PCR using PowerUp SYBR Green Master Mix (Applied Biosystems, cat. #A25742) and gene-specific primers for RPS11 (forward: 5’- GCCGAGACTATCTGCACTAC-3’ (SEQ ID NO: 147) and reverse: 5’- ATGTCCAGCCTCAGAACTTC-3’ (SEQ ID NO: 148)) and SARS-CoV-2 subgenomic N (Leader forward: 5’-GTTTATACCTTCCCAGGTAACAAACC-3’ (SEQ ID NO: 149) and N reverse: 5’-GTAGAAATACCATCTTGGACTGAGATC-3’ (SEQ ID NO: 150)). SARS-CoV-2 primers targeting genomic N (forward: 5’-TAATCAGACAAGGAACTGATTA-3’ (SEQ ID NO: 151) and reverse: 5’-CGAAGGTGTGACTTCCATG-3’(SEQ ID NO: 152)) are from Chu et a!., 2020. The following PCR conditions were used: 50 °C for 2 min and 95 °C for 2 min (initial denaturation); 45 cycles 95 °C for 1 sec, 60 °C for 30 sec (PCR); followed by 95 °C for 15 sec, 65 °C for 10 sec, a slow increase to 95 °C (0.07 °C/sec) for a melt curve. The data were analyzed by melt curve analysis for product specificity as well as AACT analysis for fold changes (after normalization to housekeeping genes) and graphed using Prism 8 (GraphPad).
Imaging of fluorescent reporters
Fluorescent and brightfield images were taken with Nikon Eclipse TE300 fluorescent microscope at xlO magnification, using NIS-Elements 4.10.01 software (Nikon).
Flow cytometry
Flow cytometry was performed on a minimum of 10,000 single cells/sample using LSRII Flow cytometer (BD Biosciences). Data analysis was done using FloJo software (BD Biosciences).
Antiviral compounds
AM580 was purchased from Cayman Chemical (#15261), Remdesivir and Masitinib were purchased from MedChemExpress (#HY- 104077 and #HY- 10209 respectively), 27- hydroxycholesterol (27HC) was purchased from Sigma Aldrich (#SML2042), Human IFN Alpha A (Alpha 2a) and Human IFN Beta (la) were purchased from Pbl assay science (#11100-1 and #11410-2 respectively).
VSV-G trans yackasins
BHK-21 cells were transfected with VSV-G or control plasmid, using Lipofectamine 3000 (ThermoFisher #L3000001), using a reverse-transfection protocol. 24h post transfection, 6 million cells were electroporated with 5ug replicon and 2ug N protein mRNA as detailed above. Each three electroporation reactions were combined into a T175 flask. Medium was replaced after a few hours overnight to remove free-floating RNA and dead cells. 48h post electroporation, supernatants were collected, filtered through a 0.45um low-protein bind filter, and concentrated xlOO by ultracentrifugation on a 20% sucrose cushion (Beckman SW28 rotor, 3h at 27,000rpm) or alternatively by overnight incubation with a final concentration of 8% PEG6000 in 0.4M NaCl followed by centrifugation at 3000g for 30min. Pellets were resuspended in TNE buffer and stored at -80C.
The work with packaged replicons was performed under biosafety level 3 conditions, in accordance with the Rockefeller University IBC committee.
Statistical Analyses
Statistical tests were used as indicated in the figure legends. Generation of plots and statistical analyses were performed using Prism8 (Graphpad). Error bars represent standard deviation unless otherwise noted. Student’s t-test (unpaired, two-tailed) was used to assess significance between treatment and control groups and to calculate P values. P < 0.05 was considered statistically significant.
EXAMPLE 2
Replicon design and optimization of RNA production
Based on extensive and analogous prior work on related coronaviruses (7(5), the SARS- CoV-2 genome is thought to encode 16 non-structural proteins (nspl-nspl6), as well as four structural proteins-spike (S), membrane (M), envelope (E), nucleocapsid (N)-and eight accessory proteins (3a, 3b, 6, 7a, 7b, 8b, 9b and 14) expressed from sub-genomic RNAs (FIG. 1A) (77). To generate SARS-CoV-2 replicons, a modulatory design was adopted to assemble two versions: a “minimal” replicon consisting of viral 5’ and 3’UTRs, Orfla/b, and N encoding regions, and a “full” replicon consisting of all viral proteins with the exception of spike (S). In both replicons, the spike transcription-regulating sequence (TRS) was used to drive expression of a gene cassette consisting of neomycin-resistance (NeoR) and a reporter gene (nuclear-localized NeonGreen, or secreted Gaussia luciferase) separated by a T2A self-cleaving sequence (FIG. 1A). Both versions contain an upstream T7 promoter for in vitro transcription at the 5’ end and a self-cleaving HDV ribozyme at the 3’ end, which cleaves immediately after an encoded polyA sequence.
For replicon assembly, an RNA virus reverse genetics system in yeast was utilized (18). This system leverages transformation-associated recombination (TAR) to efficiently and accurately assemble numerous, large overlapping DNA fragments (19). After transforming yeast with equimolar ratios of replicon fragments and confirming proper assembly with multiplex PCR, restriction digests of the resulting DNA were performed to determine plasmid integrity. Using a spike deleted NeonGreen reporter SARS-CoV-2 replicon for optimization, yeast-derived plasmids were contaminated with genomic DNA and did not reveal the expected Ndel digest pattern (FIG. IB). To circumvent this, plasmid safe (PS) DNAse treatment was used to remove contaminating yeast genomic DNA. Additionally, yeast assembled plasmids were propagated in bacteria, which considerably boosted plasmid purity, as demonstrated previously (18). However, in both instances, overall DNA yield for transcription was suboptimal and, in the case of bacterial propagation, resulted in mutations that arose mostly in the coding region of the viral RdRp, likely a major source for bacterial toxicity. To help overcome this, multiple displacement amplification (MDA) using phi29 DNA polymerase (20) and random primers were used to directly amplify plasmid preps from either yeast or sequence-verified bacterial clones. Typically used for whole genome amplification studies (21), phi29 can successfully amplify long sequences (~70kb) with high fidelity. It was reasoned that phi29 amplified replicon plasmids would thus make suitable T7 transcription templates. Indeed, amplification of replicon plasmids from yeast or bacteria yielded high amounts of full length and intact replicon DNA with no added variation (FIG. IB).
For all conditions above, the resulting DNA was used for T7 transcription reactions, and full-length replicon RNA in addition to shorter non-specific bands were observed (FIG. 1C). It should be noted an overall higher proportion of full-length replicon RNA using relative to yield using two commonly used RNA synthesis kits. To directly compare launch efficiency, these various RNAs were electroporated into BHK-21 cells together with In-vitro transcribed N protein mRNA, as described (18), and the reporter NeonGreen was measured 24h post electroporation. Notably, less than 1% of replicon positive cells using RNA from non-phi29 amplified templates with bacteria propagated plasmids yielding the highest NeonGreen reporter signal were observed (FIG. ID). In contrast, phi29 amplified templates yielded between 5-20% NeonGreen positive cells where the signal was clearly dependent on viral RdRP activity (FIG. IE). These results broadly demonstrate optimal and routine SARS-CoV-2 replicon launch from either yeast or bacteria-derived replicon plasmids after MDA (optimal schema is illustrated in FIG. IF). Stability, translation and cytotoxicity profiles of transfected RNA have been shown to be improved by 5’ cap modifications. To potentially increase the efficiency of replicon launch, three different 5’ capping strategies were tested: the co-transcriptional cap analog ARCA, post-transcriptional vaccinia virus CapO structure, and post-transcriptional vaccinia virus Cap 1 structure. All three strategies yielded similar proportions of replicon-positive cells (FIG. 1G).
Spike-deleted SARS-CoV-2 replicons are convenient and flexible assay platforms
To broadly characterize spike-deleted replicon performance as a platform for antiviral compound screening, host factor validation, and viral mutant phenotypes in an easy-to-use assay, a secreted Gaussia luciferase version, similar to previous effects with HCV reporters, was constructed (22). RNA transcripts were electroporated into Huh7.5 cells, and reporter activity was cumulatively monitored via luciferase readings from the culture supernatant. Luciferase activity was observed as early as 18 hours post-electroporation, peaking at 48 hours and observable to four days (FIG. 2A). Importantly, no luciferase signal was observed for a pol-mutant under the same conditions, and the wild-type replicon was sensitive to 100 nM of remdesivir, a well-characterized inhibitor for the SARS-CoV-2 RdRp (23). As a luciferase independent measure of replicon activity, sgRNA levels for N and M genes were measured, and similar production kinetics as the reporter gene was observed (FIG. 2B). These results suggest that SARS-CoV-2 replicons undergo sustained RNA replication in a reliable and detectable manner.
The described replicons can be used as drug screening platforms. Luciferase reporter replicons were treated with various drugs reported to have anti-coronavirus activity at different stages of the viral life cycle: the SARS-CoV-2 RdRp inhibitor remdesivir (23), a proposed 3CLpro inhibitor masitinib (25), and as a negative control, 27-hydroxycholesterol, recently reported to have both anti-SARS-CoV-2 and anti-HCoV-OC43 activity and thought to affect viral entry (26). For remdesivir, similar IC50 values and low cytotoxicity profiles as experiments with live virus were observed (FIG. 2C) (23). For masitinib, SARS-CoV-2 inhibition with attendant cellular toxicity was observed (FIG. 2D). For 27-hydroxycholesterol, as expected, no detectable inhibition was observed (FIG. 2E). These results showcase the utility of SARS-CoV-2 replicons as drug discovery platforms with a particular emphasis on intracellular replication events.
Next, whether replicons could be used to validate putative SARS-CoV-2 host factors was tested. As a proof-of-principle, TMEM41B, a critical pan-coronavirus host factor, was used (27). Replicon launch in TMEM41B KO Huh7.5 cells resulted in a 22-fold decrease in reporter activity compared to parental Huh7.5 cells (FIG. 2F), an effect with a similar magnitude to remdesivir treatment. As a control, a Sindbis virus replicon was unaffected in TMEM41B KO cells versus parental cells (FIG. 2G). Lastly, upon reconstitution of KO cells with TMEM41B, an 11-fold rescue in replicon activity was observed. These results demonstrate that SARS-CoV-2 replicons are sensitive to loss of the required host factor.
In addition, SARS-CoV-2 replicons can be used to characterize viral mutants. Recent reports have highlighted the importance of Nspl as a potent mediator of host translation shutoff (28, 29). It was reasoned that an Nspl-mutant would either permit host translation to enable sustained replicon activity or, given the proposed role of Nspl in countering the innate immune response (28), could result in a less fit replicon. Surprisingly, while Nspl- mutant replicons performed similarly to wild-type versions and could be readily inhibited by remdesivir (FIG. 21), little to no cell toxicity for Nspl -replicons was observed (FIG. 2J). Additionally, cell growth for wild-type replicons remained inhibited despite replicon launch in the presence of remdesivir (FIG. 2J). These results suggest that SARS-CoV-2 Nspl is a primary mediator of cell toxicity and, in this context, may circumvent therapeutic intervention.
SARS-CoV-2 replicons reveal viral determinants of interferon sensitivity
That Nspl -mutants were viable over five days with minimal effects on cellular toxicity led us to consider the impact of Nspl on host translation of the antiviral response. Conceptually, as Nspl activity is proposed to halt translation and therefore prevent production of ISGs (28), it was reasoned that Nspl -mutant replicons would be more sensitive to interferon. Indeed, upon replicon launch in Huh7.5 cells, Nspl-mutants were markedly sensitive to IFNa and IFN compared to wild-type replicons (FIGS. 3A-B). In contrast, both replicons exhibited similar sensitivity to remdesivir (FIG. 3C). These results highlight how Nspl subverts the antiviral ISG response and contributes to cell toxicity by halting translation.
In addition to Nspl, numerous SARS-CoV-2 accessory proteins have been proposed to inhibit cellular innate immune responses to infection (16). As the spike-deleted or “full” replicon contains the entire complement of accessory proteins, it was sought to compare this replicon to a “minimal” replicon lacking structural and accessory proteins (FIG. 3D). As shown in FIG. 3E, minimal replicon reporter has an activity that was -2-10 fold higher than the full replicon and could be monitored for up to one week and inhibited with remdesivir. A cell growth arrest phenotype with both replicons was observed (FIG. 3F). Noting the slight decrease in remdesivir sensitivity for the minimal replicon, both replicons across ranges of drug concentrations were tested. While a two-fold decrease in sensitivity for the minimal replicon with remdesivir was observed (FIGS. 5A-B), treatment with IFNa or IFNP shows similar inhibition profiles for both replicons (FIGS. 5C-F). The retinoid derivative AM580, reported to have anti-MERS activity (57), was also tested. It was found that this compound has similar inhibition profiles with both replicons. These data showcase how both full and minimal replicons perform similarly in Huh-7.5 cells.
Spike-deleted SARS-CoV-2 replicons can be packaged in trans to generate singlecycle trans-packaged replicons (TPRs)
While electroporation of replicons worked robustly in BHK-21 and Huh-7.5 cell lines, more complex cultures, such as primary cells and organoid cultures, present a challenge for efficient replicon launch. The use of such cultures is critical for studying SARS-CoV-2 biology and testing antiviral compounds in a more native context. In contrast to the minimal replicon, the full spike-deleted replicon contains the remaining E and M structural proteins. Prior work with SARS-CoV had shown that E and M proteins were sufficient to form virus-like particles (REF). It was, therefore, reasoned that launching a full SARS-CoV-2 replicon with a heterologous viral entry protein expressed in trans could yield single-cycle infectious virions that, in effect, would serve as replicon delivery particles. To test this, the vesicular stomatitis virus (YSV) G protein, commonly used to pseudotype lentivirus vectors, was expressed along with spike-deleted SARS- CoV-2 NeonGreen replicon RNA in BHK-21 cells (FIG. 4A). After filtering and concentrating the resulting supernatant, naive Huh7.5 cells were transduced, and Neongreen positive cells in a VSV-G dependent manner were observed (FIGS. 4B-C). Using these trans-packaged replicons (TPRs), several additional cell types relevant for SARS-CoV-2 were transduced: African green monkey VeroE6 cells, human Caco2 intestinal epithelial cells (32, 33), and Calu3 as well as A549 human lung adenocarcinoma cells (34-37). Additionally, transduction was tested in two lines of primary human airway cells: Normal human bronchial/tracheal epithelial cells (NHBE) and normal human lung fibroblast cells (NHLF) (FIG. 4B). Remarkably, all cells were susceptible to transduction and exhibited viral replication, as evident from NeonGreen reporter expression. Moreover, a 10-fold dilution of packaged replicons resulted in a roughly 10-fold reduced percentage of Neon Green positive Huh7.5 cells (Fig 4C), indicating the packaged replicons behave as single-cycle viral particles that can be readily titered and quantified.
To test the feasibility of the system for testing antiviral treatments, Gaussia luciferase TPRs, and transduced NHBE, NHLF, and A549 cells were generated and pretreated with remdesivir or IFNa (FIG. 4D). Both treatments markedly inhibited Glue expression, similar to what was observed with replicon RNA electroporation.
The ability of the packaged replicons to transduce lung organoids was also tested. These organoids contain several cell types organized in a complex 3 -dimensional structure, physiologically mimicking human lungs. It is a highly relevant system and an invaluable tool to study SARS-CoV-2 biology and antivirals. Organoids were constructed and transduced with NeonGreen trans-packaged replicons. Remarkably, NeonGreen signal was detected in organoids 24h post transduction (FIG. 4E) in cells expressing SOX, demonstrating that SARS-CoV2 trans- packaged replicons can transduce lung organoids.
For biological safety reasons, VSV-G RNA is provided in trans, and as a result, recombination of VSV-G RNA into the replicon is highly unlikely, as VSV-G RNA lacks the TRS sequences required to mediate the recombination events within coronavirus genomes. As a test for such events in the system, the supernatant from Huh7.5 cells was collected, concentrated, and transduced with either the Glue replicon or Glue TPRs, and attempted to transduce naive cells. While the producer cells gave a high Glue signal in a dose-dependent manner, no signal was detected in the naive cells (FIG. 4E), indicating that packaged replicons are indeed a single-cycle infection system, where no infectious particles are created in the transduced cells.
DISCUSSION This example describes the design and production of functional SARS-CoV-2 replicons. An RdRp dependent signal was observed for either fluorescent or luciferase reporter versions in multiple cell types. The RdRp dependent signal was observable for up to seven days. This example uncovered roles for the viral protein nspl as a cell-autonomous factor that limits replicon and cell viability. A role for NMD as a barrier to efficient replicon launch was also uncovered. Lastly, single cycle trans-packaged replicons were engineered as a demonstration for genome delivery in a range of cell types.
Viral replicon systems are important tools for research and as platforms for antiviral compound screening efforts. For viruses that require biosafety level 3 containment, such as SARS- CoV2, replicon systems are invaluable, as they enable studies outside of a BSL3 setting and widen the breadth of research efforts to contribute to the understanding of this pandemic-causing virus.
Compared to replicons of smaller RNA viruses, such as HCV and coronaviruses, coronavirus replicons present additional challenges due to the large size of the viral genome. This challenge is two-fold: one, genome construction is technically cumbersome, requiring in vitro ligation of DNA fragments propagated in bacteria, or via the slow, stepwise construction of low copy bacterial artificial chromosomes (BAC). The second challenge is the synthesis of a high- quality, full-length replicon RNA and its efficient introduction into cells. To address the first challenge, this example employs a yeast-based reverse genetics system to accurately assemble overlapping RNA fragments of a SARS-CoV-2 replicon into a yeast plasmid in a single transformation reaction.
Further, synthesis of full-length RNA requires sufficient amounts of high-quality DNA template. Bacterial propagation was limited to small cultures with low-copy plasmid replication, due to toxicity of viral sequences. Unfortunately, plasmid extraction from yeast contained high levels of contaminating genomic DNA. This DNA was partially removed with a plasmid-safe exonuclease, leaving the replicon plasmid intact. However, using either yeast or bacterial systems, plasmid yield was barely sufficient for the synthesis of RNA amounts required for delivery by electroporation. By utilizing MDA to amplify plasmid from these low yield preparations, this example overcame this obstacle and enabled production of large amounts of pure DNA template for in-vitro transcription. RNA synthesized with amplified DNA templates was efficiently launched in BHK-21 cells, reaching 20% reporter-positive cells. Turning to Huh-7.5 cells electroporated with the Gaussia luciferase-expressing replicon, a series of experiments were performed to demonstrate the utility of the replicon as a platform for evaluating antiviral compounds, validating a putative host factor, and characterizing viral variants. It was shown that replicon reporter activity was sensitive to remdesivir and masitinib, which inhibit the viral RdRp and the 3 CL protease, respectively, but not to 27HC, which was reported to interfere with viral entry.
TMEM41B was recently identified by the inventors in a genome-wide CRISPR screen as a host factor required for SARS-CoV2 infection (27). Remarkably, replicon reporter activity was also sensitive to loss of TMEM41B and rescued by TMEM41B reconstitution. These data validate TMEM41B as a critical host factor, but also, as replicon activity does not depend on viral entry nor particle release, further demonstrating its role on the intracellular steps of the viral life cycle.
Nspl is the first gene translated from SARS-CoV2 genomic RNA. It is also known to bind the 40S ribosome and induce a selective shutoff of the host translation, eventually leading to cell death. When electroporating replicon RNA into cells, massive cell death was observed, even though replicon launch was limited to less than 20% of the cells. The same phenotype was observed with pol(-) replicons and remdesivir-treated cells, indicating that replication is not required for cell death. It was hypothesized that Nspl, which can be translated even from partial- length incoming RNA, is responsible for this phenomenon. Indeed, when a replicon impaired in Nspl ribosome binding was used, cell viability was restored. Interestingly, Glue signal was similar to that of a WT replicon, suggesting that Nspl -mediated host shutoff is not required for basic viral replication under normal conditions. However, Nsplmut replicon was significantly more sensitive to IFNa and IFNP treatments, highlighting the role of host-shutoff in blocking IFN signaling. These data are in line with many previous publications and stress the utility of the replicon system to study the functional genome of SARS-CoV2.
The full replicon contains all viral main, structural, and accessory genes, excluding the Spike coding sequence. Previously published Coronavirus replicon studies suggest that the structural and accessory genes are not required for basic viral replication in cells. A “mini- replicon” containing only Orfla/b, N protein, and the reporter cassette was also constructed. As expected, the minireplicon efficiently launched in electroporated cells and gave a strong Glue signal. Next, the effects of different antiviral treatments on full and mini replicons were compared. While Remdesivir had a similar inhibition profile on both replicons, the minireplicon was somewhat more susceptible to PTMb treatment. Several accessory proteins are known to block different steps in IFN signaling, and future studies utilizing the mini and the full replicons with addition, deletion or mutation of specific accessory genes, would be of much interest to study their function.
A significant advantage of the full replicon as compared to minireplicon is the expression of most structural genes, which enables it to be trans-packaged. The full NeonGreen and Glue replicons were successfully trans-complemented with VSV-G protein, generating single-round infectious particles. The main advantage of using VSV-G as opposed to Spike is the wide host range of this glycoprotein. The receptor for VSV is the LDL receptor and additional members of this family (40), which also mediate infection with Lentiviruses pseudotyped with VSV-G. The LDL receptor family members are abundantly expressed in the vast majority of cell lines and primary cells from different species.
The VSV-G pseudotyped replicons efficiently transduced many cell-lines, primary cells, and lung organoids, highly relevant for SARS-CoV-2 studies. Importantly, even cell lines such as A549, which normally need overexpression of Ace2 and TMPRSS2 for SARS-CoV2 infection, were readily susceptible to transduction with NeonGreen and Glue pseudotyped replicons.
Replicon pseudotyping has many advantages over RNA electroporation or transfection of cells. For one, it can be easily titered and used in high-throughput experiments, in which cells can be pre-treated with either dose-curves of drugs or depletion or overexpression of multiple host genes. Moreover, cell electroporation involves large amounts of Nspl -encoding partial-length RNA, which, together with the harsh conditions of the electroporation, has a strong effect on cell viability. Even though the toxicity effects can be accounted for, not all cells are susceptible to electroporation or transfection, especially complex cultures such as organoids, the gold standard for testing antiviral treatments. Pseudotyped replicon assays are more efficient, reaching a higher percentage of reporter-expressing cells while having little overall toxic effect. This method delivers the viral RNA in a more physiological manner, suitable for primary cells and even whole organoids. The fact that VSV-G is expressed in trans from a separate DNA plasmid should prevent the formation of a VSV-G expressing SARS-CoV-2 virus. Thus, transduced cells are prevented from generating new infectious particles. When the concentrated supernatant of GLuc TRP- transduced cells was added onto naive cells, no GLuc signal was detected in the supernatant nor the presence of protein N in the cells by immunofluorescence, which suggests that this is a true single-cycle transduction system. Since Coronavirus recombination requires TRS sequences, it is highly unlikely that in the absence of TRS, VSV-G coding sequence would integrate into the replicon genome.
EXAMPLE 3 This example describes the materials and methods used in EXAMPLE 4.
Replicon fragment clonins
Table 1 contains all the primers used for fragment cloning. DNA fragments 2-6,8 were the same as described in Thao etal. (Thi Nhu Thao et al. , 2020). The rest of the fragments were PCR- amplified from SARS-Cov2 clone 3.1 (Thi Nhu Thao et al. , 2020), except fragment 7, which was amplified from cDNA obtained by RT-PCR of viral RNA extracted from isolate USA-WA1/2020 (BEI #NR-5281), grown in Vero-E6 cells. RNA was extracted from cells using Trizol (ThermoFisher #15596026) and RNeasy mini kit (Qiagen #74104), and cDNA was prepared using Superscript™ IV First-Strand Synthesis System with random primers (Thermo #18091050).
PCR amplification reactions were performed using KOD Xtreme Hot Start DNA Polymerase (EMD Millipore #71975). Accessory sequences, such as Neon Green, Glue, NeoR, were amplified from plasmids or purchased as synthetic DNA (IDT). PCR amplicons of fragments 1, 9, 10, 11, 12, 13 were phosphorylated with T4 PNK (NEB #M0201) and ligated directly into pGEM3Z plasmid linearized via PCR (T4 DNA ligase, NEB #M0202T). Fragment 7 pol(-) was similarly cloned into pACNR-2015FLYF\17D (Sanchez-Velazquez et al. , 2020), a low-copy plasmid. SARS-Cov2 N protein was amplified by PCR to include poly A sequence at the 3’ end and was cloned into Kpnl+Xbal digested PGEM3Z plasmid using Gibson assembly.
Fragments-containing pUC, pUC mini, and PGEM3Z plasmids were grown in E. coli DH5a (ThermoFisher), pACNR and pCCl-BAC-His3 were grown in E.coli MC1061. Plasmid DNA was extracted with ZymoPURE II Plasmid Maxiprep (Zymo research #D4202). Mutagenesis of SARS-Cov2 nspl and nspl2 was done using a two-step PCR method on fragments 2 and 7, respectively, using the primers listed in Table 1. Pol(-) mutant was created by mutating SARS-Cov2 RdRp (nspl2) D760, D761 catalytic residues to N760, N761 (SDD/SNN) (Gao et al, 2020). Nspl mutant that does not bind the 40S ribosome was created by mutating K164A/H165A (Schubert et al. , 2020; Thoms et al, 2020). The mutated fragments were used for replicon assembly, as detailed below.
Midi-replicon was constructed using alternative fragment 11, which was designed to contain only E and M genes and their regulatory sequences, but not any of the accessory genes. The primers for the EM-only fragment 11 are listed in Table SI .
Yeast assembly and bacterial propagation of assembled replicons
DNA fragments for assembly were prepared by restriction digestion or PCR as detailed in Table 2, and agarose gel extracted. Yeast assembly was performed according to the protocol in Thao et al. , 2020 (Thao et al. , 2020). Briefly, 50-100 ng of each fragment was mixed together in an equimolar ratio and transformed into Saccharomyces cerevisiae (S. cerevisiae) strain VL6-48N (ATCC #MYA-3666). Transformed yeast was grown for 2-3 days on selective -HIS plates at 30C. 4-10 colonies from each plate were picked, re-streaked on a new plate, and grown for 2 days at 30°C. Cmde DNA extraction was performed using glass beads (Biospec #11079110) and Chelex® (SigmaAldrich #95577-100G-F) and screened by multiplex PCR with the primers detailed in Table 1, using multiplex PCR kit (Qiagen #206143) according to the manufacturer's protocol. Fragment 7, which contains the coding sequence for the viral RdRp, was amplified by PCR from the positive clones and sequence-verified by sanger sequencing.
For large-scale plasmid extraction from yeast, the protocol in Thao et al. , 2020 (PMID: 32833212) was used as described, except ZymoPURE II Plasmid Maxiprep (Zymo research #D4202) was used instead.
To eliminate yeast chromosomal DNA, the plasmid prep was digested with BamHI-HF enzyme (NEB #R3136T), which does not have restriction sites in any of the pCCl-BAC-His3- replicon plasmids. Next, DNA was digested with Plasmid-Safe™ ATP -Dependent DNase (Lucigen #E3101K) for 24h and cleaned by extraction with Phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation. Final DNA concentration was measured using Qbit dsDNA HS Assay (ThermoFisher #Q32851) For bacterial propagation, lpl of the crude yeast DNA prep was electroporated into E. coli TransforMax™ Epi300™ electrocompetent cells (Lucigen #EC300110). The bacteria were then plated on chloramphenicol-containing plates, and grown at 30°C overnight. Bacterial clones were used to make 5ml starters cultures and grown overnight at 30°C. The following day, the starter was diluted 1:10 in fresh LB+Choramphenicol with copy-control solution (Lucigen #CCIS125), and incubated in a shaker incubator at 37°C for 5h. Plasmids were then extracted using Qiaprep spin miniprep kit (Qiagen #27104). As with the yeast preps, fragment 7 was amplified by PCR sequence-verified by sanger sequencing.
Multiple displacement amplification (MPA) of replicon plasmids and DNA template preparation.
For amplification of pCCl-BAC-His3-replicon plasmids, 5-30ng of DNA was used with EquiPhi29™ DNA Polymerase (ThermoFisher #A39390) according to manufacturer’s instructions, using exonuclease-resistant random primers (ThermoFisher #S0181).
Amplified DNA was digested with Notl-HF enzyme (NEB #R3189S), and cleaned up with phenol-chloroform-isoamylalcohol (SigmaAldrich #77617), followed by ethanol precipitation. The resulting DNA was sequence verified in full by amplicon high-throughput sequencing at the Harvard MGH CCIB DNA Core Facility.
In vitro transcription and electroporation ofRNA
Transcription reactions were set up with lpg linear DNA templates using HiScribe™ T7 High Yield RNA Synthesis kit (NEB #E2040S) or T7 RiboMAX™ Express Large Scale RNA Production System (Promega #P1320). After in-vitro transcription, RNA was treated with Ambion™ DNase I (ThermoFisher #AM2222) and cleaned using Monarch® RNA Cleanup Kit (NEB #T2050L). For co-transcriptional capping ofRNA, Anti-Reverse Cap Analog (ARCA) was used at 1:2.8 (GTP:ARCA) ratio (NEB #S 1411 S). For post-transcriptional capping, CellScript ScriptCap™ Cap 1 Capping System was used with ScriptCap™ 2'-0-Methyltransferase to generate Capl, or without ScriptCap™ 2'-0-Methyltransferase to generate CapO. Concentration and quality was measured by nanodrop. To visualize the size of the RNA product, RNA was loaded on 0.8% agarose gel and Gel Loading Buffer II (ThermoFisher #AM8546G).
Sindbis replicon RNA was in-vitro transcribed from a SinRep5-GFP plasmid linearized with Xhol (NEB) (Bredenbeek et al. , 1993), using SP6 mMessage mMachine High Yield Capped RNA Transcription kit (ThermoFisher #AM1340) RNA transcripts were electroporated into Huh-7.5 or BHK-21 cells using adapted protocols originally developed for launching HCV (Lindenbach et cil, 2005). Briefly, Huh-7.5 or BHK-21 cells were trypsinized, washed twice with ice-cold phosphate-buffered saline (PBS) (Invitrogen), and resuspended at 1.5 x 107 cells/ml in PBS. Subsequently, 5pg of SARS-CoV-2 replicon RNA and 2pg of SARS-CoV2 N mRNA were mixed with 0.4ml of cell suspension in a 2-mm cuvette (BTX #45-0125) and immediately pulsed using a BTX ElectroSquare Porator ECM 830 (860V, 99 ps, five pulses). Electroporated cells were incubated at room temperature for 10 min prior to resuspension in plating media.
Cell culture
Huh-7.5 cells (H. sapiens ; sex: male) (Blight et al, 2002), Caco-2 (ATCC® HTB-370™, H. sapiens ; sex: male), A549 (ATCC® CCL-185™, H. sapiens ; sex: male) and VeroE6 cells (ATCC® CRL-1586™, Chlorocebus sabaeus ) were maintained at 37° °C, and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Fisher Scientific, cat. #11995065) supplemented with 0.1 mM nonessential amino acids (NEAA, Fisher Scientific, cat. #11140076) and 10% hyclone fetal bovine serum (FBS, HyClone Laboratories, Lot. #AUJ35777). BHK-21 cells (ATCC CCL-10, M. auratus) were grown in Minimum Essential Medium (MEM, Fisher Scientific, cat. #11095080), supplemented with 8% FBS and 0.1 mM NEAA. Calu-3 cells (ATCC® HTB-55™, H. sapiens ; sex: male) were grown in Eagle's Minimum Essential Medium (EMEM, ATCC® 30- 2003™) supplemented with 10% FBS and 0.1 mM NEAA on collagen-coated dishes. Normal Human Lung Fibroblasts (NHLF, Lonza #CC-2512) and Normal Human Bronchial Epithelial cells (NHBE, Lonza #CC-2541) were subcultured using Reagent Pack™ Subculture Reagents (Lonza #CC-5034) and maintained in FGM™-2 Fibroblast Growth Medium-2 BulletKit™ (Lonza #CC- 3132) and BEGM™-2 BulletKit™ Medium (Lonza #CC-3170) respectively.
TMEM41B-KO and dox-inducible TMEM4 IB-reconstituted Huh-7.5 cells were previously described (Hoffmann etal, 2020). TMEM41B expression was induced by Doxycycline (MilliporeSigma #D9891) at least 24h before electroporation.
Huh-7.5 ACE2+TMPRSS2 cells were made by transduction with lentivirus bearing a TMPRSS2-2A-NeoR_ACE2 csssette (Schneider WM et al, 2020) and selection with G418 (500pg/ml).
All cell lines have tested negative for contamination with mycoplasma.
Antiviral compounds AM580 was purchased from Cayman Chemical (#15261), Remdesivir and Masitinib were purchased from MedChemExpress (#HY- 104077 and #HY- 10209 respectively), 27- hydroxycholesterol (27HC) was purchased from Sigma Aldrich (#SML2042), Human IFN Alpha A (Alpha 2a) and Human IFN Beta (la) were purchased from Pbl assay science (#11100-1 and #11410-2 respectively).
Secreted gaussia luciferase assays
Following electroporation, between 15-30K cells in IOOmI media per well were plated into 96 well plates containing equal volumes (IOOmI) of indicated compound dilutions at 2x concentrations. Cell supernatants were cumulatively harvested at various timepoints or at 24hrs post-electroporation and luciferase signal was measured using the Renilla luciferase assay system (Promega, E2820) in a Fluostar Omega microplate reader (BMG Labtech). Cell viability was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega #G7570) following the manufacturer's instructions. aPCR
Huh-7.5 cells containing repSARS-CoV-2 Glue, minirepSARS-CoV-2 Glue, orrepSARS- CoV-2 Glue pol- replicons were seeded onto 12-well plates in triplicate at 1 x 105 cells/well and treated with lOOnM remdesivir or DMSO vehicle. After incubating for 24 or 48hours at 37° °C, supernatants were aspirated, cells were washed three times with PBS and subsequently lysed in 250 mΐ Tri-reagent (Zymo, cat. #R2050) per well. RNA was extracted using the Direct-zol RNA Miniprep Plus kit (Zymo Research, cat. #R2072) according to the manufacturer's protocol, followed by reverse transcription into cDNA using random hexamer primers with the Superscript III First-Strand Synthesis System Kit (Invitrogen, cat. #18080051) following the manufacturer’s instructions. Gene expression was quantified by qRT-PCR using PowerUp SYBR Green Master Mix (Applied Biosystems, cat. #A25742) and gene-specific primers for RPS11 (forward: 5’- GCCGAGACTATCTGCACTAC-3 ’ (SEQ ID NO: 147) and reverse: 5’- ATGTCCAGCCTCAGAACTTC-3’ (SEQ ID NO: 148)) and SARS-CoV-2 subgenomic N (Leader forward: 5’-GTTTATACCTTCCCAGGTAACAAACC-3’ (SEQ ID NO: 149) and N reverse: 5’-GTAGAAATACCATCTTGGACTGAGATC-3’ (SEQ ID NO: 150)). SARS-CoV-2 primers targeting genomic N (forward: 5’-TAATCAGACAAGGAACTGATTA-3’ (SEQ ID NO: 151) and reverse: 5 ’ -CGAAGGTGTGACTTCC ATG-3 ’ (SEQ ID NO: 152)) are from Chu et al., 2020. The following PCR conditions were used: 50 °C for 2 min and 95 °C for 2 min (initial denaturation); 45 cycles 95 °C for 1 sec, 60 °C for 30 sec (PCR); followed by 95 °C for 15 sec, 65 °C for 10 sec, a slow increase to 95 °C (0.07 °C/sec) for a melt curve. The data were analyzed by melt curve analysis for product specificity as well as AACT analysis for fold changes (after normalization to housekeeping genes) and graphed using Prism 8 (GraphPad).
Immunofluorescence
For immunofluorescence experiments, between 15-30K cells in IOOmI media per well were plated into black- walled clear bottom 96-well plates (Coming, cat. #3904) following replicon electroporation or TPR transduction. At the time of harvest, cells were prepared by fixation with 2% paraformaldehyde (PFA) for at least 20 minutes. PFA was then removed, and cells were resuspended in PBS+1%FBS and stored at 4°C until processed. Cells were washed in PBS containing 0.1% Tween-20 (PBST) and permeabilized with PBS containing 0.1% Triton X-100 for 10 min at room temperature. After washing with PBST, cells were incubated for 1 hour at room temperature with a blocking solution of 5% BSA in PBST. To stain SARS-CoV-2 infected cells, a rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (GeneTex, cat. #GTX135357) was added to the cells at 1:1000-2000 dilution in PBST. VSV-Gwas stained with a mouse monoclonal anti-VSV-G antibody isolated from II hybridoma supernatant (ATCC CRL-2700) as described (Schmidt et al. , JEM, 2020) and) used at 1:2000 in PBST. Spike was stained using the human C144 monoclonal antibody described in (Robbiani et al, 2020) and at 1:2500 dilution in PBST. After overnight incubation at 4°C, cells were washed and stained with secondary antibodies donkey anti-rabbit AlexaFluor 594 (ThermoFisher Scientific, cat. #A-21207) at 1:2000, donkey anti mouse AlexaFluor 594 (Abeam, cat. #abl 50108) at 1:2000, or donkey anti-human DyLight 594 (ThermoFisher Scientific, cat. #SAS-10128) at 1:500. Nuclei were stained with DAPI (ThermoFisher Scientific, cat. #D1306) at 1 pg/ml, prior to imaging. For the results in Figure 1 and Figure 5D, fluorescent and brightfield images were taken with a Nikon Eclipse TE300 fluorescent microscope at lOx magnification, using NIS-Elements 4.10.01 software (Nikon). For the results in Figures 4, 5, S3, and S5, fluorescent images were obtained on a Keyence BZ-X710 microscope at 4x magnification, and quantified on Neon and antibody stained signals (N, VSV-G and/or Spike) using the Hybrid Cell Count tool in the BZ-X Analyzer software (version 1.3.03).
Flow cytometry
Cells were prepared by fixation with 2% paraformaldehyde (PFA) for at least 20 minutes. PFA was then removed, and cells were resuspended in PBS+1%FBS. Flow cytometry was performed on a minimum of 10,000 single cells/sample using LSRII Flow cytometer (BD Biosciences). Data analysis was done using FloJo software (BD Biosciences).
Spike and VSV-G trans-packasim
BHK-21 cells were transfected with Spike or VSV-G (plasmid sequence available upon request) plasmid using Lipofectamine 3000 (ThermoFisher #L3000001), using a reverse- transfection protocol. 24h post transfection, 6 million cells were electroporated with 5ug replicon and 2ug N protein mRNA as detailed above. Cells were either seeded in a collagen-coated (Collagen from calf skin, Sigma #C8919) T75 flask for one electroporation, or 3 electroporations were combined into a single T175 flask. For P0 imaging, some of the electroporated cells were seeded in parallel on collagen-coated 96 wells plates for imaging. The supernatant was collected 24h post electroporation for Spike TPRs or 48h for VSV-G TPRs, and cleared by centrifugation. TRP concentration was done by overnight incubation of the supernatant with a final concentration of 8% PEG6000 in 0.4M NaCl at 4°C followed by centrifugation at 3000g for 30min. Pellets were resuspended in TNE buffer and stored at -80°C.
Quantification and statistical analysis
Statistical tests were used as indicated in the Figure legends. Generation of plots and statistical analyses were performed using Prism8 (Graphpad). Error bars represent standard deviation, unless otherwise noted. Student’s t-test (unpaired, two-tailed) was used to assess significance between treatment and control groups, and to calculate P values. P < 0.05 was considered statistically significant.
EXAMPLE 4
Replicon design and optimization of RNA production
Based on prior work on related coronaviruses, the SARS-CoV-2 genome is thought to encode 16 non- structural proteins (nspl-nspl6) in two overlapping reading frames (Orfla/lb), as well as four structural proteins - Spike, membrane (M), envelope (E), nucleocapsid (N) - and eight accessory proteins (3a, 3b, 6, 7a, 7b, 8b, 9b, and 14) expressed from sub-genomic RNAs or alternative reading frames. To generate SARS-Cov-2 replicons, a modular design was adopted to assemble two versions: a “minimal” replicon consisting of viral 5’ and 3’ElTRs, Orfla/b, and N encoding regions and a “full” replicon consisting of all viral proteins with the exception of the primary structural glycoprotein Spike (AS). In both replicons, the Spike transcription-regulating sequence (TRS) was used to drive expression of a gene cassette consisting of neomycin-resistance (NeoR) and a reporter gene (nuclear-localized NeonGreen or secreted Gaussia luciferase) separated by a T2A self-cleaving sequence (FIG. 1A). Both replicon versions contain an upstream T7 promoter for in vitro transcription at the 5’ end and a self-cleaving hepatitis delta virus (HDV) ribozyme at the 3’ end, which cleaves immediately after an encoded polyA sequence.
For replicon assembly, an RNA virus reverse genetics system in yeast was used. This system leverages transformation-associated recombination (TAR) to efficiently and accurately assemble numerous, large overlapping DNA fragments. After transforming yeast with equimolar ratios of replicon fragments and confirming proper assembly with multiplex PCR, restriction digests of the resulting DNA were performed to determine plasmid integrity. Using a AS NeonGreen-encoding SARS-CoV-2 replicon for optimization, it was consistently observed that yeast-derived plasmids were contaminated with genomic DNA and did not reveal the expected Ndel digest pattern (FIG. IB). To circumvent this, a plasmid safe (PS) DNAse treatment was utilized to remove contaminating yeast genomic DNA. Additionally, yeast assembled plasmids were propagated in bacteria, which considerably boosted plasmid purity. However, the overall DNA yield for transcription was suboptimal in both instances and, in the case of bacterial propagation, resulted in mutations in the coding region of the viral RNA-dependent RNA polymerase (RdRp), likely a major source for bacterial toxicity. To help overcome this, multiple displacement rolling circle amplification (RCA) using phi29 DNA polymerase and random primers were used to directly amplify replicon plasmids from either yeast or sequence-verified bacterial clones. RCA generally amplifies circular DNA with high selectivity over residual chromosomal DNA or PS DNAse digestion products due to greater efficiency during the initial phase of amplification. Due to its processivity (>70kb per binding event), strand-displacement activity, and low error rate (<1E'6), phi29 polymerase can exponentially amplify long, circular DNA sequences with high fidelity under isothermal conditions and has been used to replicate bacterial artificial chromosomes, cosmids, mitochondrial DNA, and microbial genomes up to megabases in length. It was reasoned that phi29 amplified replicon plasmids would thus make suitable T7 transcription templates. Indeed, amplification of replicon plasmids from yeast or bacteria yielded high amounts of full-length and intact replicon DNA with no added variation (FIG. IB). For all conditions above, the resulting DNA was used for T7 transcription reactions, and full-length replicon RNA in addition to shorter non-specific bands was observed (FIG. 1C). An overall higher proportion of full-length replicon RNAs was also observed relative to yield using two commonly used RNA synthesis kits (FIG. 5B). To directly compare launch efficiency, these RNAs were electroporated into BHK-21 cells together with in vitro transcribed N mRNA, and the NeonGreen reporter was measured 24h post electroporation. Notably, less than 1% of replicon positive cells was observed using RNA from non-phi29 amplified templates, with bacteria propagated plasmids yielding the highest NeonGreen reporter signal (FIG. ID). In contrast, phi29- amplified templates yielded between 5-20% NeonGreen positive cells in a viral RdRP activity- dependent manner (FIG. IE). These results demonstrate that optimal and routine SARS-CoV-2 replicon launch can be achieved using yeast or bacteria derived replicon plasmids after RCA (optimal scheme is illustrated in FIG. IF). Stability, translation, and cytotoxicity profiles of transfected RNA have been shown to be improved by 5’ cap modifications. To potentially increase the efficiency of replicon launch, three different 5’ capping strategies were tested: the co- transcriptional cap analog ARCA and post-transcriptional vaccinia virus Cap 0 and Cap 1 structures. All three strategies yielded similar proportions of replicon positive cells, with a slight advantage to post-transcriptional capping (FIG. 1G).
Spike-deleted SARS-CoV-2 replicons are convenient and flexible assay platforms
To broadly characterize Spike-deleted replicon performance as a platform for antiviral compound screening, host factor validation, and viral mutant phenotypes in an easy-to-use assay, a secreted Gaussia luciferase (Glue) version was constructed (FIG. 1A). RNA transcripts were electroporated into Huh-7.5 cells, and cumulative reporter activity was monitored via luciferase readings from culture supernatant. Luciferase activity as early as 18 hours post-electroporation, peaking at 48 hours, and persisting for additional days was observed (FIG. 2A). The activity of luciferase was dependent on viral replication, as no signal was observed for an RdRP mutant (pol- ) under the same conditions, and the wild-type replicon was sensitive to remdesivir, a well- characterized inhibitor of the SARS-CoV-2 RdRp (Pruijssers el ah, 2020). As a luciferase independent measure of replicon activity, subgenomic RNA (sgRNA) levels were measured for N and observed similar production kinetics as the reporter gene (FIG. 2B) compared to input N mRNA measurements. These results show that SARS-CoV-2 replicons undergo sustained RNA replication in a robust and quantifiable manner.
The disclosed replicons can be used as drug screening platforms. Cells bearing luciferase reporter replicons were treated with compounds reported to have anti-coronavirus activity at different stages of the viral life cycle: the SARS-CoV-2 RdRp inhibitor remdesivir (Pruijssers l al, 2020) and a proposed 3CLpro inhibitor masitinib (Drayman etal. , 2020). As a negative control, 27-hydroxy cholesterol was used, which was reported to have both anti-SARS-CoV-2 and anti- HCoV-OC43 activity by affecting viral entry. For remdesivir, IC50 values and low cytotoxicity profiles similar to those previously reported for live viruses were observed (FIG. 2C). For masitinib, SARS-CoV-2 inhibition with concomitant cellular toxicity was observed (FIG. 2D). Also, 27-hydroxycholesterol showed no detectable inhibition (FIG. 2E). Additionally, a hosttargeting agent (HTA), AM580, a retinoid derivative reported to have broad antiviral activity by disrupting sterol regulatory element binding protein (SREBP) lipid signaling was also tested. For AM580, IC50 values were consistent with reported results (N. Drayman et al, bioRxiv 2020.08.31.274639 (2020)), with low cytotoxicity at inhibitory concentrations. These results demonstrate the utility of SARS-CoV-2 replicons as scalable drug discovery platforms with a focus on intracellular replication events.
Next, whether replicons could be used to validate putative SARS-CoV-2 host factors was tested. To this end, the transmembrane protein 41B (TMEM41B), a critical host factor for multiple coronaviruses (Schneider et al. , 2020) was used. Replicon launch in TMEM41B KO Huh-7.5 cells resulted in a 22-fold decrease in reporter activity compared to wild-type (FIG. 2F), an effect with a similar magnitude to remdesivir treatment. In contrast, a Sindbis virus replicon was not affected by TMEM41B deletion (FIG. 2G). Lastly, TMEM41B reconstitution in KO cells led to an 11-fold rescue in replicon activity (Figure 2H). These results demonstrate that SARS-CoV-2 replicons are sensitive to disruption of critical intracellular host factors.
SARS-CoV-2 replicons can also be used to characterize viral mutants. Recent studies have highlighted the importance of Nspl as a potent mediator of host translation shutoff (Lapointe et al. , 2021; Schubert et al. , 2020; Thoms et al. , 2020), (Tidu et al. , 2020),(Baneijee et al. , 2020). It was reasoned that an Nspl- mutant would either permit host translation to enable sustained replicon activity or could result in a less fit replicon. Surprisingly, while Nspl- mutant replicons performed similarly to wild-type versions and could be readily inhibited by remdesivir (FIG. 21), little to no cellular toxicity for Nspl- replicons was observed (FIG. 2J). Additionally, cell growth for wild-type replicons remained low despite replicon launch in the presence of remdesivir (FIG. 2J). These results suggest that SARS-CoV-2 Nspl is a primary mediator of cell toxicity that may circumvent therapeutic intervention in this context.
SARS-CoV-2 replicons reveal viral determinants of interferon sensitivity The fact that Nspl -mutants were viable over five days with minimal effects on cellular toxicity led to consideration of the impact of Nspl on host translation of the antiviral response. As Nspl activity is proposed to halt translation and therefore prevent production of Interferon- stimulated genes (ISGs), it was hypothesized that Nspl - mutant replicons would be more sensitive to interferon. Indeed, upon replicon launch in Huh-7.5 cells, it was observed that Nspl- mutants were markedly sensitive to IFNa and IFNP compared to wild-type replicons (FIGS. 3A-B). In contrast, both replicons exhibited similar sensitivity to remdesivir (FIG. 3C). These results highlight how Nspl subverts the antiviral ISG response and contributes to cell toxicity by impacting translation. More broadly, these data highlight the modularity of the replicon platform for systematic studies aimed at dissecting viral phenotypes driven by different proteins.
In addition to Nspl, numerous SARS-CoV-2 accessory proteins have been proposed to inhibit cellular innate immune responses to infection. Next, the AS replicon, which contains the entire complement of accessory proteins, was compared to a “minimal” replicon lacking structural and accessory proteins. The minimal replicon produced -2-10 fold higher reporter activity than the AS replicon, could be monitored for up to one week, and was also sensitive to remdesivir. Limited cell growth was observed with both replicons. Given a slight decrease in remdesivir sensitivity of minimal replicon, a range of drug concentrations were tested for both replicons across. A two-fold decrease in sensitivity for the minimal replicon with remdesivir and a sevenfold increase in sensitivity to IFNa, but not IFNp were observed. The retinoid derivative AM580, reported to have anti-MERS activity, was also tested, and similar inhibition profiles for both replicons were observed. These data demonstrate that both AS and minimal replicons are functional in Huh-7.5 cells and can be used for probing the function of specific viral components.
SARS-CoV-2 replicons can be trans-complemented with Spike to generate singlecycle virions
While BHK-21 and Huh-7.5 cells are permissive to replicon launch via electroporation, other more relevant lung cell lines and primary cell types are not as amenable. Further, electroporation largely precludes the study of entry processes. As an alternative route for replicon delivery, it was next sought to package replicons as single-cycle virions that would infect cells in a Spike-dependent manner but would not make infectious progeny. To accomplish this, it was reasoned that Spike expression in trans could package the AS replicon if expressed in the same cell. While in principle similar to pseudotyped lentiviruses, these single-cycle virions were referred to as trans-packaged replicons (TPRs) to highlight the emphasis on replicon delivery. To implement this, BHK-21 cells were transfected with a Spike expressing plasmid, and 24 hours later AS NeonGreen replicon RNA was electroporated (FIGS. 7A-B). The Spike gene cassette is human-codon optimized and contains none of the 5’ leader sequence, TRS sequence, or 3’UTR that would mediate potential recombination. In addition, as Nspl hinders cell survival post electroporation, it was further hypothesized that Nsp 1 -deficient replicons might be packaged more efficiently compared to wild-type replicons. To test this, supernatant harboring wild-type or Nspl- TPRs was concentrated and used to transduce recipient Huh-7.5 cells overexpressing ACE2 and TMPRSS2 (Huh-7.5 AT). NeonGreen positive cells in a Spike-dependent manner were readily detected (FIGS. 7C-D PI), with Nspl- TPRs yielding higher reporter activity. Upon passing the supernatant from transduced cells onto naive Huh-7.5 AT cells, no NeonGreen positive cells were detected (FIGS. 7C-D P2), suggesting that TPRs are single-cycle infectious. Consistent with this, Spike expression was readily detected in the producer cells by immunostaining, but it did not carry over to the PI cells (FIGS. 8A-B). In contrast, N protein expression was observed in producer and PI cells, but not in P2 cells (FIG. 8C). Further consistent with a single-cycle infectivity, Spike TPR transduction does not yield characteristic syncytia formation indicative of Spike expression, as is typically the case with live viruses (FIGS. 8D-E). These results broadly demonstrate that SARS-CoV-2 replicons can be trans-complemented with Spike to produce virions with singlecycle infectivity.
Neutralization assays with TPRs recapitulate authentic SARS-CoV-2 antibody phenotypes
With the emergence of SARS-CoV2 Spike variants across the globe, how TPRs could provide an isogenic SARS-CoV-2 based platform upon which single-cycle virions with differing Spikes could be constructed and tested in neutralization assays was investigated. This would complement existing assays performed using pseudotyped and chimeric lenti- or rhabdoviruses as a CoV-based single cycle alternative. To test this, Nsplmut-Gluc TPRs trans-packaged with either the wild-type Spike (WA1/2020 isolate) or the B.1.351 South African variant of concern were generated. Neutralization assays were performed with these TPRs using two monoclonal antibodies previously tested against these SARS-CoV-2 variants in pseudotype and infectious virus assays (Weisblum et al. , 2020). Previous work with pseudotyped and chimeric viruses bearing wild-type or B.1.351 Spikes demonstrated that while C135 efficiently neutralizes both variants, C144 neutralizes the wild-type, but not the B.1.351 variants (Robbiani l al.. 2020; Weisblum et al, 2020). Similar phenotypes were observed using wild-type and B.1.351 Spike TPRs (FIGS. 7E-F). C135 and C144 were capable of potently neutralizing SARS-CoV-2 or pseudoviruses expressing the prototype spike. C144 binding and neutralization is ablated by the spike E484K mutation, which was originally identified by antibody selection experiments using a VSV pseudovirus and which later appeared in the B.1.351 variant. TPRs harboring the prototype spike were neutralized with both antibodies, while only C135, but not C144, neutralized B.1.351 spike TPRs (FIGS. 7E-F and FIGS. 9A-C). Neutralization curves and relative IC50 values were comparable to those obtained with the respective SARS-CoV-2 isolates and with previous reports using pseudovirus assays (D. F. Robbiani, e/ al. Nature 584, 437-442 (2020); Y. Weisblum, etal eLife 9, e61312 (2020)) with the added advantage that TPR generation does not require deletions in the spike coding sequence (F. Schmidt, etal J. Exp. Med. 217, e20201181 (2020)). Moreover, neutralization curves and IC50 values were comparable to those obtained with infectious SARS- CoV2 viruses, where N staining was used to quantify positive cells (FIGS. 9A-C). These data demonstrate that Spike-deleted replicons can function as a rapid and modular platform to generate single-cycle virions with variant Spikes.
SARS-CoV-2 replicons can be packaged with VSV glycoprotein
Efficient transduction with Spike TPRs requires high levels of ACE2. Since numerous cell lines relevant for SARS-CoV-2 studies have insufficient ACE2 levels unless engineered otherwise to support viral entry, we next sought to overcome this limitation for replicon launch. Trans complementation was tested using the glycoprotein from vesicular stomatitis virus (VSV-G), as it is commonly used to pseudotype lentiviral vectors for a wide range of applications. BHK-21 cells were transfected with a VSV-G expression plasmid followed by electroporation with AS NeonGreen replicon RNA. After concentrating the resulting supernatant, naive Huh-7.5 cells were transduced, and NeonGreen positive cells in a VSV-G dependent manner were observed. Importantly, there was no measurable signal in a second passage, and no VSV-G carryover was detected in the PI cells, demonstrating that the VSV-G TPRs are single-cycle infectious. In contrast to the Spike TPRs, producer (P0) VSV-G expressing cells had a significantly higher ratio of NeonGreen compared to control cells when the Nspl- replicon was used, suggestive of local VSV-G dependent spread. PI cells also exhibited higher NeonGreen and N protein positivity when transduced with Nspl - TPRs compared to wild-type TPRs. VSV-G TPR infectivity was tested on additional cell types relevant for SARS-CoV-2: African green monkey VeroE6 cells, human Caco2 intestinal epithelial cells (Letko et cil, 2020; Verhoeckx el ah, 2015), and both Calu3 and A549 human lung adenocarcinoma cells (Blanco-Melo etal., 2020; Cagno, 2020; Giard etal. , 1973; Zhu et al, 2010). Additionally, TPR transduction was tested in two lines of primary human airway cells: normal human bronchial/tracheal epithelial cells (NHBE) and normal human lung fibroblast cells (NHLF). All cells were susceptible to transduction with VSV-G TPRs and exhibited viral replication, as evident from NeonGreen reporter expression.
To further demonstrate that VSV-G TPRs are single-cycle infectious using a more sensitive approach, Glue expressing VSV-G TPRs were generated, and naive Huh-7.5 cells were transduced followed by Glue measurements 24h later (PI). While the VSV-G TPR-transduced cells produced a robust Glue signal, no such signal was observed when the supernatant from PI was concentrated and passed onto naive cells (P2). Input supernatant was similarly concentrated and used as positive control for VSV-G TPR concentration and transduction.
Next, VSV-G TPR performance was tested in primary or ACE2 deficient contexts for antiviral compound screening. NHBE, NHLF, and A549 cells were pre-treated with lOOnM remdesivir or lOOpM IFNa and subsequently transduced these cells with Glue VSV-G TPRs. Both treatments markedly inhibited Glue reporter activity to levels approaching baseline. Overall, these data demonstrate that VSV-G TPRs provide a single-cycle and flexible means of replicon launch.
VSV-G trans-complementation of a SARS-CoV-2 replicon lacking all accessory genes
As TPR results thus far have relied on full Spike-deleted replicons with or without mutations in Nspl, if replicons harboring larger deletions could be packaged was tested. Initially, it was observed that the minimal replicon is unable to be packaged using VSV-G. This expected result reflects the requirement for M and E genes to promote virion morphogenesis. A replicon that lacks all accessory genes but retains genes encoding the structural M and E proteins (AAcc) was next generated and tested packaging with VSV-G. Comparing WT and AAcc VSV-G TPRs, similar VSV-G dependent infectivity was observed on recipient Huh-7.5 cells using both NeonGreen and Glue versions. These results demonstrate that accessory genes are dispensable for replicon packaging with VSV-G.
DISCUSSION
The above examples describe a novel SARS-CoV-2 replicon platform that is functional across a range of primary and established cell lines from various tissue types. Replicon constmction is modular, utilizing yeast-based recombination and whole genome amplification methods to reliably and rapidly assemble multiple variant SARS-CoV-2 replicons. It was demonstrated that the replicon signal is RdRp-dependent, sensitive to antiviral compounds across multiple cell types in a dose-dependent manner, and can be used to validate intracellular host factors such as TMEM41B (Schneider et al, 2020). Further, a role for the Nspl viral protein in limiting replicon and cell viability in a cell-autonomous manner and as a key arbiter for sensitivity to interferons was uncovered. Leveraging these features, single-cycle virions packaged with either Spike or VSV glycoproteins for efficient replicon delivery was generated. The former permits detailed one-way studies of SARS-CoV-2 entry and replication; while the latter permits replicon delivery that is not limited by a lack of ACE2 expression.
Compared to replicons of smaller RNA viruses, such as HCV and alphaviruses, coronavirus replicons present two main challenges due to the large size of the viral genome. First, genome construction is technically cumbersome, requiring in vitro ligation of DNA fragments propagated in bacteria or via slow, stepwise construction of low copy bacterial artificial chromosomes (BACs). The second challenge is the synthesis of a high-quality full-length replicon RNA and its efficient introduction into cells. These challenges were address herein by pairing yeast recombination to assemble replicon plasmids with a Phi29 DNA polymerase amplification step to ensure sufficient DNA for in-vitro transcription.
The resulting RNA was efficiently launched in BHK-21 and Huh-7.5 cells, reaching up to 20% reporter-positive cells following electroporation. We initially attributed the lack of higher efficiencies to the difficulty of generating replication competent full-length RNA. However, the considerable cell death observed upon electroporation, even with RdRP deficient replicons or wild- type replicons treated with remdesivir, prompted us to focus on Nspl. As the first gene translated from SARS-CoV2 genomic RNA, Nspl is known to bind the 40S ribosome and induce a selective shutoff of the host translation (Schubert et al. , 2020). It was hypothesized that Nspl, which could be translated even from partial-length incoming RNA, might be impacting cell survival. Using an Nspl mutant unable to engage the 40S ribosome, cell viability was restored without grossly affecting the replicon. Reporter activity for Nspl- replicons was similar to wild-type replicons, indicating that Nspl-mediated host shutoff is not required for viral replication in this context. Further, the Nspl - replicon was significantly more sensitive to interferons, highlighting the role of host-shutoff in blocking IFN signaling, as has been previously reported (Tidu et al, 2020; Xia et al. , 2020),(Banerjee el al. , 2020). Overall, these data highlight the utility of replicon systems as platforms to study probe SARS-CoV-2 protein function.
Reverse genetics systems for SARS-CoV-2 that permit single-cycle virion production have thus far relied on trans-complementation with the N gene (Ju etal, 2021) or with the Orf3a and E genes (Zhang et al. , 2021a). With both strategies, single-cycle infectivity is efficiently achieved by deleting an essential structural component of the virion from the viral genome and supplying it in Irons. The strategy to trans-complement with Spike operates under a similar principle, but has several distinct potential advantages. First, a single Spike-deleted replicon can be packaged with variant Spikes, without replicon re-engineering. This provides a modular, rapid, and isogenic means of generating single-cycle virions for Spike-directed efforts, such as screening variants against neutralizing antibodies. Second, single-cycle infectivity may also be achieved with VSV- G, analogous to one-way lentivirus transduction. This sidesteps the requirement for ACE2, which may be beneficial for studies in model systems where ACE2 overexpression is not feasible. And finally, the full complement of viral proteins are functionally available in cis for detailed study in a Spike-deleted replicon, should trans-complementation not fully recapitulate some viral phenotypes.
Part of the inability to observe virus rescue may be explained by purposefully employed strategies to address the specific risk of recombination. For one, the Spike and VSV-G expression plasmids contain none of the 5 TJTR, TRS, or 3 TJTR sequences that are required for discontinuous transcription by the viral replicase. This helps to ensure that Spike or VSV-G mRNAs are ill-suited templates for the viral RdRP. While the constructs for Spike and VSV-G are human codon- optimized for maximal expression, proposed approaches to strategically codon de-optimize Spike presents an additional future means of attenuation. Second, replicons harboring loss-of-function mutations in virulence-associated genes such as Nspl may help ensure that any rescued virus is debilitated. The decrease in cell death observed with Nspl- TPRs, combined with the increased sensitivity to interferons, is consistent with this idea. Along similar lines, replicons harboring deletions of multiple accessory proteins may provide an additional layer of attenuation, given that CoV accessory proteins are dispensable for replication in cell culture, but often impact pathogenesis in vivo (Liu et al. , 2014). The observation that replicons lacking all accessory genes can be packaged serves as a platform to test this idea, and in addition, provides a single-cycle context to explore accessory protein function in mediating virulence. Overall, SARS-CoV-2 Spike- deleted replicons and TPRs provide a flexible and non-infectious platform for future studies of this pandemic virus.
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Claims

CLAIMS What is claimed is:
1. A nucleic acid molecule comprising or encoding a coronavirus replicon that comprises:
(i) a genomic or subgenomic nucleotide sequence of a coronavirus, wherein the nucleotide sequence comprises at least one of the coding sequences of a membrane (M) protein of the coronavirus and an envelope (E) protein of the coronavirus and wherein the coding sequence of a spike (S) protein of the coronavirus is inactivated or deleted; and
(ii) a second nucleotide sequence encoding a selectable marker suitable for selection, wherein the selectable marker is under the control of the RNA virus replication machinery.
2. The nucleic acid molecule of claim 1, wherein the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
3. The nucleic acid molecule of any one of the preceding claims, wherein the selectable marker is a gene that confers resistance to an antibiotic.
4. The nucleic acid molecule of any one of the preceding claims, further comprising a reporter gene.
5. The nucleic acid molecule of claim 4, wherein the reporter gene is selected from the group consisting of NeonGreen, gaussia luciferase (Glue), mScarlet, green fluorescent protein (GFP), blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Venus, mOrange, Topaz, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), HcRed, t-HcRed, DsRed, DsRed2, t-dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP,
Monster GFP, paGFP, Kaede protein, a Phycobiliprotein, and a biologically active variant or fragment of thereof.
6. The nucleic acid molecule of any one of the preceding claims, wherein the coding sequence of the S protein or a portion thereof is replaced with the second nucleotide sequence.
7. The nucleic acid molecule of any one of claims 4 to 6, wherein the reporter gene is operatively linked to a spike transcription-regulating sequence (TRS).
8. The nucleic acid molecule of claim 7, wherein the reporter gene is operatively linked to the spike transcription-regulating sequence (TRS) through a T2A self-cleaving sequence.
9. The nucleic acid molecule of any one of the preceding claims, wherein the nucleic acid molecule has at least 80% sequence identity to SEQ ID NO: 1 or 3, or comprises the nucleic acid sequence of SEQ ID NO: 1 or 3.
10. A virus particle or a virus-like particle comprising the nucleic acid molecule of any one of the preceding claims.
11. The virus particle or virus-like particle of claim 10 comprising a vesicular stomatitis virus G (VSV-G) protein.
12. A cell or cell line comprising the nucleic acid molecule of any one of the preceding claims.
13. The cell or cell line of claim 12, further comprising a second nucleic acid molecule comprising a coding sequence of a vesicular stomatitis virus G (VSV-G) protein or a variant thereof.
14. The cell or cell line of claim 13, wherein the vesicular stomatitis virus G (VSV-G) protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 2 or comprises the amino acid sequence of SEQ ID NO: 2.
15. The cell or cell line of claim 12, further comprising a third nucleic acid molecule comprising a coding sequence of a Spike protein or a variant thereof.
16. The cell or cell line of claim 15, wherein the Spike protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4 or comprises the amino acid sequence of SEQ ID NO: 4.
17. The cell or cell line of any one of claims 12 to 16, wherein the cell is a Huh-7 cell or derived from a Huh-7 cell.
18. The cell or cell line of claim 17, wherein the cell is a Huh-7.5 cell.
19. The cell or cell line of any one of claims 12 to 16, wherein the cell is in a lung organoid.
20. A composition comprising the nucleic acid molecule of any one of claims 1 to 8 or the cell or cell line of any one of claims 12 to 19, and a pharmaceutically acceptable carrier.
21. A kit comprising the nucleic acid molecule of any one of claims 1 to 8, the cell or cell line of any one of claims 12 to 19 or the composition of claim 20.
22. A method of preparing a coronavirus replicon-harbored cell, comprising: (i) introducing to a cell the nucleic acid molecule of any one of claims 1 to 8; and
(ii) maintaining the coronavirus replicon in the cell in a cell culture medium containing an antibiotics to which the selection marker confers resistance.
23. The method of claim 22, wherein the cell comprises a second nucleic acid comprising a coding sequence of the vesicular stomatitis virus G (VSV-G) protein or a variant thereof.
24. The method of claim 22, wherein the cell comprises a third nucleic acid comprising a coding sequence of a Spike protein or a variant thereof.
25. The method of any one of claims 22 to 23, wherein the cell is a Huh-7 cell or derived from a Huh-7 cell.
26. The method of claim 25, wherein the cell is a Huh-7.5 cell.
27. A method for screening for antiviral agents for a coronavirus, comprising:
(i) contacting the cell or cell line of any one of claims 12 to 19 with a candidate agent; and
(ii) determining an increase or decrease in replication or activity of the coronavirus virus replicon relative to a control cell or cell line harboring the same replicon, wherein the control cell or cell line has not been contacted with the candidate agent.
28. The method of claim 27, wherein the step of determining comprises determining a level of production of a coronavirus protein or a coronavirus RNA transcript or the marker or the reporter.
29. The method of any one of claims 27 to 28, wherein the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
30. The method of any one of claims 27 to 29, wherein the candidate agent comprises an organic compound or an antisense nucleic acid.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050130127A1 (en) * 2001-05-17 2005-06-16 Rottier Petrus J.M. Coronavirus-like particles comprising functionally deleted genomes

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US20050130127A1 (en) * 2001-05-17 2005-06-16 Rottier Petrus J.M. Coronavirus-like particles comprising functionally deleted genomes

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FERNANDES ET AL.: "Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses", VIRUSES, vol. 12, no. 6, 2020, pages 598, XP055883449, DOI: https://doi.org/10.3390/v12060598 *
RICARDO-LAX INNA, LUNA JOSEPH M., THAO TRAN THI NHU, LE PEN JÉRÉMIE, YU YINGPU, HOFFMANN H.-HEINRICH, SCHNEIDER WILLIAM M., RAZOOK: "Replication and single-cycle delivery of SARS-CoV-2 replicons", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 374, no. 6571, 26 November 2021 (2021-11-26), US , pages 1099 - 1106, XP055956151, ISSN: 0036-8075, DOI: 10.1126/science.abj8430 *

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