WO2023108299A1 - Polypeptides capable of limiting the replication of a coronavirus - Google Patents

Polypeptides capable of limiting the replication of a coronavirus Download PDF

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WO2023108299A1
WO2023108299A1 PCT/CA2022/051850 CA2022051850W WO2023108299A1 WO 2023108299 A1 WO2023108299 A1 WO 2023108299A1 CA 2022051850 W CA2022051850 W CA 2022051850W WO 2023108299 A1 WO2023108299 A1 WO 2023108299A1
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nucleic acid
coronavirus
acid molecule
isolated nucleic
cells
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PCT/CA2022/051850
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French (fr)
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Gerard Pelletier
Samer GIRGIS
Ioannis RAGOUSSIS
Spyridon OIKONOMOPOULOS
Silvia VIDAL
Danielle Malo
Tom HOBMAN
Zaikun XU
Pavel Baranov
Alla FEDOROVA
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The Royal Institution For The Advancement Of Learning/Mcgill University
The Governors Of The University Of Alberta
University College Cork - National University Of Ireland, Cork
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Publication of WO2023108299A1 publication Critical patent/WO2023108299A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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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
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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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
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention pertains to polypeptides and/or nucleic acids encoding polypeptides that are capable of attenuating coronavirus replication and methods of using same for treating coronavirus infections.
  • Coronaviruses are a highly diverse family of enveloped positive single-stranded RNA viruses that cause diseases in mammals and birds. They are divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. The seven human coronaviruses have been identified and they all belong to the genera alphacoronavirus or betacoronavirus. These seven coronaviruses primarily cause respiratory symptoms in humans that range from mild to lethal. Gammacoronavirus and deltacoronavirus have no known viruses that infect humans, but contain important agricultural pathogens of livestock.
  • Coronaviruses possess remarkably large RNA genomes, which range in size from 27 to 33kb and are flanked by 5’ and 3’ untranslated regions that contain cis-acting secondary RNA structures that are essential for RNA synthesis.
  • the genome of a coronavirus is organized as follows: a 5’-leader-untranslated region (UTR) - Open reading frames (ORF) 1a and 1b - spike (S) - envelope (E) - membrane (M) - nucleocapsid (N) - 3’UTR-poly(A) tail.
  • the ORFs 1a and 1 b which occupy the first two-thirds of the genome, encode two large overlapping polyproteins, pp1a and pplab.
  • the larger polyprotein, pplab is a result of a -1 ribosomal frameshift site at the end of open reading frame ORF1a.
  • the ribosomal frameshift allows for the continuous translation of ORF1a followed by ORF1 b.
  • the polyproteins pp1a and pplab have their own proteases that cleave the polyproteins at different sites. The cleavage of polyprotein pplab yields 16 nonstructural proteins (Nsp1 - Nsp16).
  • Nsps 2-16 compose the viral replication and transcription complex that includes, amongst others, RNA-processing and RNA-modifying enzymes and a RNA proofreading function necessary for maintaining the integrity of the coronavirus genome.
  • the ORFs that encode the viral major structural proteins (S, E, M and N) and interspersed ORFs that encode accessory proteins are transcribed from the 3’ one-third of the genome to form a nested set of subgenomic mRNAs. While ORF1a/ORF1ab and the four canonical structural proteins (S, E, M and N) are common to all coronaviruses, the number of accessory proteins and their function is unique depending on the specific coronavirus.
  • the SARS-CoV-2 genome which is -29.9 kB in size, encodes a total of 27 different proteins.
  • Coronavirus infections begin when the spike protein attaches to its complementary host cell receptor. After attachment, a protease of the host cell cleaves and activates that receptor-attached spike protein which allows the virus to fuse with the host cell. The release of the coronavirus genome into the host cell cytoplasm upon entry marks the onset of a complex program of viral gene expression, which is highly regulated in space and time. Translation of ORF1a and ORF1 by the host translational machinery leads to the production of the pp1a and pp1 b polyproteins, which are subsequently cleaved to into the individual Nsps.
  • Nsps assemble into the replicase-transcriptase complex (RTC) to create environment suitable for RNA synthesis and are ultimately responsible for RNA replication and transcription of subgenomic RNAs.
  • the Nsps also contain other enzyme domains and functions, including those for RNA replication.
  • Nsp1 promotes cellular mRNA degradation and blocks host cell translation and Nsp12 encodes the RNA- dependent RNA polymerase.
  • the viral structural proteins, S, E and M are translated and moved along to the ER-Golgi immediate compartment (ERGIC).
  • ERGIC ER-Golgi immediate compartment
  • dexamethasone have been administered systemically to severely ill Covid-19 patients in a hyper-inflammatory state.
  • systemic corticosteroids in treating severe coronavirus infections is controversial due to the side effects associated with their use.
  • corticosteroids might actually allow increased viral replication and aggravate the disease.
  • Vaccines have long been considered the gold standard for infectious disease prevention and eradication.
  • zoonotic pathogens like coronaviruses emerge from animal reservoir species, thus vaccination strategies are unlikely to lead to eradication while the virus continues to circulate in reservoir hosts.
  • Monoclonal antibodies have potential utility in combating viral diseases caused by coronavirus, by prophy lactically and therapeutically neutralizing structural proteins on the outside of the virion. Indeed, mAbs have been shown to improve survival in patients hospitalized with COVID-19 who were unable to mount effective immune responses to the virus on their own. Further, mAbs may also have a role in preventing infections, particularly in unvaccinated close contacts of individuals who are known to be infected with a coronavirus.
  • one drawback of mAb therapy is that, due to differences in the structural proteins between different coronavirus species, separate formulations of mAbs are likely to be required for different coronavirus species and/or strains. Additionally, it may be possible for coronaviruses to evolve mutations that allow them to escape neutralization by the mAb.
  • the present disclosure concerns isolated polypeptides, isolated nucleic acid molecules and/or therapeutic interfering particles derived from coronaviruses that are capable to limiting coronavirus replication.
  • the present disclosure provides an isolated polypeptide capable of limiting the replication of a coronavirus, whereby the isolated polypeptide comprises a fusion of at least two polypeptides encoded by viral open reading frames of a ORF1a of a coronavirus.
  • the two polypeptides are Nsp1 and Nsp10 and the fusion polypeptide comprises a Nsp1 moiety and a Nsp10 moiety.
  • the Nsp1 moiety of the fusion protein comprises the amino acid sequence of SEQ ID NO:3, a variant of the amino acid sequence of SEQ ID NO: 1 or 3 and/or a fragment of the amino acid sequence of SEQ ID NO: 1 or 3.
  • the Nsp10 moiety of the fusions protein comprises the amino acid sequence of SEQ ID NO:4, a variant of the amino acid sequence of SEQ ID NO: 2 or 4 and/or a fragment of the amino acid sequence of SEQ ID NO: 2 or 4.
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 5, a variant of the amino acid sequence of SEQ ID NO: 5 and/or a fragment of SEQ ID NO:5.
  • the present disclosure provides an isolated nucleic acid molecule encoding the fusion polypeptides described herein.
  • the isolated nucleic acid molecule comprises one or more ribonucleic acid residues.
  • the isolated nucleic acid molecule is a messenger RNA (mRNA) molecule. In still other embodiments, the isolated nucleic acid molecule comprises one or more deoxyribonucleic acid (DNA) residues. In yet other embodiments, the isolated nucleic acid molecule is a DNA molecule. In some embodiments, the isolated nucleic acid molecule further comprises a heterologous gene.
  • mRNA messenger RNA
  • DNA deoxyribonucleic acid
  • the isolated nucleic acid molecule is a DNA molecule. In some embodiments, the isolated nucleic acid molecule further comprises a heterologous gene.
  • the present disclosure provides an isolated nucleic acid molecule comprising a defective viral genome (DVG) encoding a therapeutic interfering particle (TIP), wherein the TIP: (i) is capable of limiting the replication of a human coronavirus, (ii) is replication defective and can be replicated in the presence of a helper virus; (iii) is defective for packaging and can be packaged in the presence of a helper virus,
  • DVG defective viral genome
  • TIP therapeutic interfering particle
  • (iv) is capable of being enriched upon a plurality of passage in a cell infected with the coronavirus and/or the helper virus at a multiplicity of infection equal to or greater than 1 ;
  • the TIP encoded in the DVG is a defective interfering particle (DIP).
  • the coronavirus is the helper virus.
  • the human coronavirus and/or the helper virus is from the alpha genus. In other embodiments, the human coronavirus and/or the helper virus is 229E or NL63.
  • the human coronavirus and/or the helper virus is from the beta genus. In further embodiments, the human coronavirus and/or the helper virus is 0043, HKU1 , SARS- CoV, MERS-CoV, or SARS-CoV2. In yet other embodiments, the human coronavirus and/or helper virus is from the gamma genus. In other embodiments, the human coronavirus and/ or helper virus is from the delta genus.
  • the isolated nucleic acid molecule comprising a defective viral genome may comprise, when compared to the nucleic acid sequence of the coronavirus or of the helper virus, a first deletion in an open reading frame 1a (ORF1a).
  • the isolated nucleic acid molecule contains a first deletion at a first start position corresponding to position 749 of GenBank accession number NC_045512.
  • the first deletion ends at the first position corresponding to position 13311 of GenBank accession number NC_045512.
  • the first deletion in an ORF1a encompasses the nucleic acid sequence encoding at least one of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, or Nsp9.
  • the TIP encoded in the nucleic acid molecule comprising a DVG is capable of limiting the titer of the coronavirus and/or helper virus by at least one log or more.
  • the isolated nucleic acid molecule has a nucleic acid sequence of GI.285 (SEQ ID NO:6), GI.249 (SEQ ID NO:7), GI.616 (SEQ ID NO:8), GI.50 (SEQ ID NO:9), GI.55 (SEQ ID NO:10) or GI. 535 (SEQ ID NO:11 ).
  • the present disclosure provides a TIP comprising the polypeptide defined in any one of the first aspect and/or the nucleic acid molecule of the second aspect.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising (i) the polypeptide of the first aspect and/ or the isolated nucleic acid molecule of the second aspect and (ii) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is formulated for nasal administration.
  • the present disclosure provides a method for treating a coronavirus infection in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the polypeptide of the first aspect, the isolated nucleic acid of the second aspect and/or the pharmaceutical composition of the fifth aspect, to the subject so as to reduce the replication of the coronavirus.
  • the subject is a human subject.
  • the present disclosure provides the use of a therapeutically effective amount of the polypeptide of the first aspect, the isolated nucleic acid of the second aspect and/or the pharmaceutical composition of the fifth aspect, for treating a coronavirus infection in a subject in need thereof.
  • the subject is a human subject.
  • the present disclosure provides the polypeptide of the first aspect, the isolated nucleic acid of the second aspect, and/or the pharmaceutical composition of the fifth aspect, for treating a coronavirus infection in a subject in need thereof.
  • the subject is a human subject.
  • Fig. 1A is schematic diagram illustrating a strategy to generate SARS-CoV-2 DIPs in Vero E6 cells.
  • Northern blotting and nanopore direct (or native) RNA sequencing were used to assess the emergence of prominent DVGs during serial passaging.
  • Fig. 1C is a photograph of a representative set of plaque assays performed on Vero E6 cells with supernatant taken at the indicated passages.
  • Fig. 1 D shows a genome coverage of nanopore RNA sequencing data from P1 , P14, and P30.
  • the “step” changes (indicated by upward arrows in P1) occur at the 5' borders of the S, 3a, E, 6, and N sgRNAs.
  • the reference genome position (nt) is shown at the bottom.
  • Fig. 1 E is a schematic diagram of the SARS-CoV-2 genome illustrating ORF organization.
  • the boxes with nucleotide coordinates and upper-case letters denote location of 32P-labelled probes (A to G) used for Northern blotting.
  • Fig. 1 F is a Northern blot analysis performed on RNA isolated from Vero E6 cells infected with SARS-CoV-2 from the indicated passages after a short exposure (S.E) (2 h/RT). Plus sign, asterisks, and arrow highlight prominent 5 kb DVGs emerging from P20- P30.
  • Fig. 1G is a Northern blot analysis performed on RNA isolated from Vero E6 cells infected with SARS-CoV-2 from the indicated passages after to long exposure (L.E.) (3.5 h/- 70°C with intensifying screen). Plus sign, asterisks, and unlabelled arrow highlight prominent 5 kb DVGs emerging from P20-P30.
  • Fig. 1 H shows genome coverage of nanopore RNA sequencing data from P1 , P15, and P29.
  • the “step” changes (indicated by upward arrows in P1 ) occur at the boundary of the S, 3a, E, 6, and N ORFs and are reflective of the 5’ borders of the major sgRNAs.
  • the reference genome position (nt) is shown at the bottom.
  • Fig. 11 shows the architecture of the top 50 most abundant DVGs from P15 infected cells, including the percentage of read counts corresponding to the transcript model.
  • the “% of Transcript Model Reads” corresponds to the number of viral reads mapped to a collapsed transcript model (as shown for individual DVGs plotted to the right) divided by the total number of all viral RNA reads x 100. Open colored boxes are retained sequences and thin lines correspond to deletions.
  • the CoV-2 reference genome is shown above the DVGs, along with the encoded polypeptides. Nucleotide position is shown below.
  • Fig. 1J shows the architecture of the top 50 most abundant DVGs from P29 infected cells, including the percentage of read counts corresponding to the transcript model.
  • the “% of Transcript Model Reads” corresponds to the number of viral reads mapped to a collapsed transcript model (as shown for individual DVGs plotted to the right) divided by the total number of all viral RNA reads x 100. Open colored boxes are retained sequences and thin lines correspond to deletions.
  • the CoV-2 reference genome is shown above the DVGs, along with the encoded polypeptides. Nucleotide position is shown below.
  • Fig. 1 K is a Northern blot analysis performed on RNA isolated from the indicated passages of SARS-CoV-2-infected cells with Nsp12.
  • Fig. 1L is a Northern blot analysis performed on RNA isolated from the indicated passages of SARS-CoV-2-infected cells with ORF10+3’UTR.
  • Fig. 1 M shows the architecture of the top seven most abundant DVGs obtained from R1 -infected cells from Exp #1 having retained 5' and 3' end sequences.
  • the SARS- CoV-2 reference genome is shown at the top, along with the encoded polypeptides.
  • Fig. 1 N shows the architecture of the top seven most abundant DVGs obtained from P14 -infected cells from Exp #1 having retained 5' and 3' end sequences.
  • the SARS- CoV-2 reference genome is shown at the top, along with the encoded polypeptides.
  • Fig. 10 shows the architecture of the top seven most abundant DVGs obtained from P30 -infected cells from Exp #1 having retained 5' and 3' end sequences.
  • the SARS- CoV-2 reference genome is shown at the top, along with the encoded polypeptides.
  • Fig. 1 P is a schematic showing the location of primers used for amplification (A1/A2) and for assessing presence of USJ (US-J/1/USJ-2) and DSJ (DSJ-3/DSJ-4) fragments in GI.535. Shown are the nucleotide sequenc es flanking the breakpoints in GI.535.
  • Fig. 1Q is an image of an end-point polymerase chain reaction (PGR) gel showing emergence of most prominent DVGs at P20 with stable maintenance to P30. Amplifications were performed using primers MIK2. Products were obtained following 30 amplification cycles and analyzed on a 0.8% agarose/TAE gel.
  • PGR polymerase chain reaction
  • Fig. 1U is a pie chart illustrating relative abundance of DVGs in P25 from Experiment 1.
  • Fig. 1V is a pie chart illustrating relative abundance of DVGs in P30 from Experiment 1.
  • Fig. 2A is a schematic showing the location of primers used for long range PGR amplification are indicated.
  • Complementary DNA was prepared using oligo d(T) and primers A1 and A2 were used in the long-range PCRs.
  • Junction primers were designed based on direct RNA nanopore sequencing and used to amplify the corresponding genomes from the long-range PCR products.
  • Fig. 2B is an image of a gel showing PCR products obtained with primers A1 and A2 using LA-Taq hot start DNA Polymerase (Takara) following long-range amplification from RNA isolated from uninfected Vero E6 cells (Mock) or Vero E6 cells infected with the indicated viral passages.
  • H 2 O sample contained no input RNA in RT reaction.
  • Fig. 2C is an image of a gel showing PCR products obtained using the indicated junction primer pair B1/B2 and the PCR product generated in Fig. 2B as template. Arrows indicate PCR products obtained of the expected molecular mass.
  • Fig. 2D is an image of a gel showing PCR products obtained using the indicated junction primer pair B1/C1 and the PCR product generated in Fig. 2B as template. Arrows indicate PCR products obtained of the expected molecular mass.
  • Fig. 2E is shematic showing the location of primers used for long range PCR amplification.
  • the difference between Gl. 616 and GI.50 is a 19 amino acid in-frame deletion in Nsp12.
  • Fig. 2F is an image of a gel of PCR products obtained following long-range amplification from RNA isolated from Vero E6 cells infected with the indicated viral passages.
  • H 2 O samples contained no input RNA in RT reaction, M (NEB 1 kb DNA ladder).
  • Arrow indicates PCR product that was gel purified and Sanger sequenced by primer walking. Primers spanning or unique to the Nsp12 internal deletion were used to distinguish GI.616 from GI.50, respectively.
  • Fig. 2G is a sequencing chromatogram showing the 5’ Nsp1/10 (750/13312 or 751/13313) junction fragment obtained by Sanger sequencing.
  • Fig. 2H is a sequencing chromatogram showing the 3’ Nsp13/N (16829/29446) junction fragment obtained by Sanger sequencing.
  • Fig. 3A shows a genome architecture of the two most prevalent DVGs present in infected Vero E6 at P15 and P29 (Exp#1 ) and at P30 (Exp #2). Nucleotide position is based on the SARS-CoV-2 Wuhan-Hu-1 isolate (NC_045512.2). Asterisks denotes a missense mutation converting 344 CTC 346 to 344 TTC 346 in Nsp1.
  • Fig. 3B shows a schematic representation of the structure of the indicated DVGs. Right angled arrows indicate location of primers used for qPCR analysis.
  • Fig. 3F is an image of a gel obtain for end-point PCR products (P2 to P8). Amplifications were performed using primers USJ-1 and DSJ-4. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
  • Fig. 3G is an image of a gel obtain for end-point PCR products (P9 to P30). Amplifications were performed using primers USJ-1 and DSJ-4. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
  • Fig. 3I is a Northern blot analysis performed on RNA isolated from the indicated SARS-CoV-2 infected cells with probe B. Arrow highlights prominent DVGs emerging at late passages.
  • Fig. 3J is a Northern blot analysis performed on RNA isolated from the indicated SARS-CoV-2 infected cells with probe G. Arrow highlights prominent DVGs emerging at late passages.
  • Fig. 3K shows the characterization of DVGs obtained in Exp#2 by nanopore direct RNA sequencing. Genome coverage of the nanopore direct RNA sequencing data from P1 , P14, and P30. The “step” changes (indicated by upward arrows in P1 ) occur at the boundary of the S, 3a, E, 6, and N ORFs and are reflective of the 5’ borders of the major sgRNAs.
  • Fig. 3L shows the architecture of the top 50 most abundant DVGs from P30 infected Vero cells. DVGs from P30 in Vero cells (Expt#2) were used. The I percentage of read counts corresponding to each transcript model is shown.
  • Fig. 3M is a schematic diagram of the SARS-CoV-2 genome illustrating ORF organization.
  • the boxes with nucleotide coordinates and upper-case letters denote location of 32P-labelled probes used for Northern blotting.
  • Fig. 3N is a Northern blot analysis performed on intracellular RNA isolated from the indicated passages.
  • RNA markers NEB
  • kb kilobases
  • the assignment of sgRNAs is based on predicted size.
  • the plus sign and asterisks highlight DVGs present in P14.
  • the same Northern blot was used in all probings shown in this panel.
  • GAPDH was used to assess mRNA quality. Mock, uninfected cells; gRNA, genomic RNA.
  • Fig. 30 is a Northern blot analysis performed on RNA isolated from SARS-CoV-2 infected cells at the indicated passages.
  • Fig. 3P shows the genome coverage of nanopore DRS data from P1 , P15 and P29.
  • the “step” changes (indicated by upward arrows in P1 ) occur at the 5’ border of the S, 3a, E, 6, and N sgRNAs.
  • Fig. 3Q shows the architecture of the top 7 most abundant DVGs from P1 infected cells obtained in Exp#2 and that had retained 5’ and 3’ end sequences, the read counts corresponding to the transcript model are also shown.
  • Fig. 3R shows the architecture of the top 7 most abundant DVGs from P15 infected cells obtained in Exp#2 and that had retained 5’ and 3’ end sequences, the read counts corresponding to the transcript model are also shown.
  • Fig. 3S shows the architecture of the top 7 most abundant DVGs from P29 infected cells obtained in Exp#2 and that had retained 5’ and 3’ end sequences, the read counts corresponding to the transcript model are also shown.
  • Fig. 3T shows the architecture of GI.616 and GI.50 in an inverted black triangle denoting a 19 amino acid deletion in GI.616. GI.50 and GI.616 which harbor identical US and DS junctions.
  • Fig. 3U is an image of end-point PGR showing emergence of most prominent DVG (P2-P8). Amplifications were performed using primers A1 and A2. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
  • Fig. 3V is an image of end-point PGR showing emergence of most prominent DVG (P9-P30). Amplifications were performed using primers A1 and A2. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
  • Fig. 3W is a pie chart illustrating the relative abundance of DVGs in P20 from Exp#2.
  • Fig. 3X is a pie chart illustrating the relative abundance of DVGs in P25 from Exp#2.
  • Fig. 3Y is a pie chart illustrating the relative abundance of DVGs in P30 from Exp#2.
  • Fig. 3Z shows the genome architecture of the most prevalent DVGs isolated from infected cells at P30. Nucleotide position is based on the SARS-CoV-2 Wuhan-Hu-1 isolate (NC_045512.2).
  • Fig. 4A is a schematic showing that DVGs are packaged and present in extracellular supernatant. Vero E6 cells were infected with virus from P1 , P16, or P30 and after 24 h, RNA was isolated from the supernatant or infected cells for analysis.
  • Fig. 4B shows RT-qPCR analysis of RNA isolated from supernatant (S/N) or the cytoplasm (Cyto) of infected cells at the indicated passages.
  • Fig. 4G is a Northern blot analysis of cellular RNA from the indicated sources and viral passages. Exposure time for the blot probing cellular RNA (lanes 1-3) was 3 days (- 70°C/intensifying screen).
  • Fig. 4D is a Northern blot analysis of S/N RNA from the indicated sources and viral passages. Exposure time for the blot probing the supernatant (S/N) RNA (lanes 4-6) was 14 days (-70°C/intensifying screen).
  • Fig. 5B is a schematic diagram showing DVG-dependency on parental virus for replication and propagation. At low MOI (0.0002) in which parental genomes and Dis enter different cells, the DI will be lost upon sequential serial passaging.
  • Fig. 5D is an image of gel from amplification products of Dis from Vero cell lysates and media that had been infected with the indicated viral stocks at an MOI of 1 or 0.0002. Amplifications were performed using A1 and A2 primers for 30 cycles. Products were analyzed on a 0.8% agarose/TAE gel. White arrows indicate recovery of 5 kb DVGs. M; 1 kb DNA ladder.
  • Fig. 6A shows an experimental flow used to generate synthetic DI particles.
  • cells were transfected with in vitro synthesized Renilla luciferase (RLuc) mRNA, GI.50, GI.55, or GI.616 RNA eight hours post-infection (hpi).
  • RLuc Renilla luciferase
  • hpi in vitro synthesized Renilla luciferase
  • Supernatant was collected 22 h later, clarified, and used to infect a new set of Vero E6 cells four subsequent times.
  • the location of the upstream junction (USJ) and the downstream junction (DSJ) primers is indicated on the map of the synthetic template.
  • Fig. 6C shows results after Vero E6 cells were transfected with H 2 O (control), GI.50, GI.55, or GI.616 and cell media was collected 22 h later, clarified and used to “infect” new cells. This was repeated one more time (P2 cells).
  • RT-qPCR analysis of RNA from P0 (transfected) and P2 cells. RNAs targeted by each oligo pair is shown on the bottom. Obtained Ct values are displayed, n 2 ⁇ SD.
  • Fig. 6D is a an image of a gel showing RT-Long range PCR showing recovery of ID genomes from P4 infected cells. RNA from the indicated samples were used to generate cDNA using random primers, followed by long range PCR amplification.
  • Fig. 6F is schematic diagram of DI genomes harboring EMCV/RLuc expression cassette.
  • Fig. 6G is a graph showing the luciferase activity obtained from cells mock transfected or transfected with Ren, GI.55-EMC/RLuc and GI.616-EMC/RLuc mRNA. Extracts were prepared from cells that had been mock infected (mock) or received SARS- CoV-2 virus. The supernatant was harvested 24 hpi (PO) or after 1 to 4 serial passages of the viral supernatant obtained from PO cells (labelled P1 - P4).
  • Fig.7C is an image of a gel showing recovery of DI genomes from SP4-infected cells.
  • Fig. 7D is a Western blot of extracts probed with a-Nsp1 or a-actin antibodies. Lysates analyzed were prepared from uninfected (mock) Vero E6 cells (lane 1 ) or Vero cells receiving SP2 from untransfected cells (-) (lane 2), RLuc mRNA-transfected cells (lane 3), or GI.616 RNA-transfected cells (lane 4). Dotted arrow denotes Nsp1 and red arrow denotes Nsp1-10 fusion.
  • Fig. 7F is a graph showing a RT-qPCR analysis of RNA from P0, SP2, and SP4 infected cells.
  • RLuc, DI USJ and DSJ, and CoV-2 gRNA RNA levels were calculated as a fold change relative to GAPDH using the 2-ACT method. Values corresponding to the USJ and DSJ junction were averaged to yield a final DI level.
  • RLuc and DI levels were expressed relative to CoV-2 gRNA levels.
  • Fig. 7I is a schematic diagram of DI genomes harboring EMCV/RLuc or TRS/RLuc expression cassettes.
  • Fig. 7L is an image of a gel of a RT-PCR showing the presence of an RLuc sgRNA containing sequences upstream of the 5' TRS-L site in SARS-CoV-2-infected cells transfected with GI.616-TRS/RLuc.
  • Fig. 8A is a schematic diagram showing the experimental design for assessing the effect of GI.616 on SARSCoV- 2 replication.
  • SARS-CoV-2 and GI.616 genomes were isolated from P2 and P3 cells, as well as SP3 supernatant.
  • Fig. 8B is a graph showing the growth rates (absolute gRNA levels relative to the amount at 4 h) of parental virus propagated in the presence of GI.616 (+) or RLuc (-). The data for two independent experiments is shown (Exp1 and Exp4).
  • Fig. 8C is a graph showing the growth rates (absolute gRNA levels relative to the amount at 4 h) of parental virus propagated in the presence of GI.616 (+) or RLuc (-). The data for two independent experiments is shown (Exp2 and Exp3).
  • Fig. 9A is schematic showing the coding potential of Dis. Black triangle indicates the 19 amino acid deletion in GI.616.
  • Fig. 9B is a Western blot of extracts probed with a-Nsp1 antibodies. Lysates analyzed were prepared from uninfected (mock) Vero E6 cells or Vero E6 cells infected with P2, P15, and P30 (Exp #2) viral stocks. Dotted arrow denotes Nsp1, filled arrow denotes arrow denotes Nsp1-10 fusion.
  • Fig. 9C is a Western blot of extracts probed with a-Nsp10 C-terminal domain antibodies. Lysates analyzed were prepared from uninfected (mock) Vero E6 cells or Vero E6 cells infected with P2, P15, and P30 (Exp #2) viral stocks. Unnbelled arrow denotes Nsp1-10 fusion.
  • Fig. 9D is an image of a gel demonstrating that Nsp1-10 is predominantly a cytoplasmic protein.
  • pcDNA-based expression vectors were transfected into 293T cells, and 48 h later cells were harvested in PBS. Following subcellular fractionation (C, cytoplasm; N, nuclear), proteins were resolved on a 10% SDS-polyacrylamide gel, transferred to immobilon PVDF membrane, and probed with antibodies indicated to the right.
  • eEF2 and hnRNPAI were used as loading controls for cytoplasmic and nuclear fractions, respectively.
  • Fig. 9E is a Clustal Omega alignment of Nsp1 and Nsp1-10 fusion. The extent of two Nsp1 deletion mutants are indicated by dotted lines. The location of the Nsp1 KH amino acids that were mutated to AA are indicated.
  • Fig. 9F is a graph showing a polysome analysis of 293 T cells transfected with the indicated expression vectors (20 pg). Cytoplasmic extracts were prepared 24 h posttransfections and polysomes analyzed by sucrose gradient sedimentation.
  • Fig. 9G is a Western blot analysis undertaken on the protein samples obtained from individual polysome fractions as per Fig. 9F. Western blots were probed with antibodies shown to the right.
  • Fig. 9H is a graph showing a polysome analysis of 293T cells transfected with FLAG-Nsp1 (KH/AA) expression vector (10 pg). Cytoplasmic extracts were prepared 24h post-transfections and polysomes analyzed by sucrose gradient sedimentation.
  • Fig. 9I is a Western blot analysis of protein samples obtained from individual fractions as per Fig. 9H. Fraction numbers are indicated. Western blots were probed with antibodies shown to the right.
  • Fig. 9J is a graph showing that the ectopic expression of Nsp1-10 does not inhibit translation in 293 T cells.
  • 293 T cells were transfected with the indicated amounts of expression vector. Twenty-four hours later, cells were metabolically labeled with 35S- Met/Cys for 15 min. TCA precipitation was used to determine the amount of radiolabel incorporated into proteins and counts were normalized to total protein content in the extract and expressed relative to cells having received empty vector (pcDNA3; which was set to 1).
  • n 4 biologically independent experiments ⁇ SD. ns, p > 0.05; *, 0.01 > p > 0.05 (Dunnett’s multiple comparisons test).
  • Fig. 9K is a Western blot analysis of protein samples obtained in Fig. 9J. Protein extracts were prepared from cells transfected with the indicated expression vectors, resolved on a 10% SDS-polyacrylamide gel, and transferred to immobilon PVDF mem branes. Western blots were probed with antibodies shown to the right.
  • Fig. 9L is a coomassie stain of 10% SDS-PAGE of purified recombinant protein used in in vitro translation assays.
  • Fig. 9M is a graph showing in vitro inhibition of translation by recombinant Nsp1 , but not Nsp1-10.
  • Fig. 90 is a graph showing a RT-qPCR analysis of RNA from SP4 infected cells.
  • the DI USJ and DSJ, and CoV-2 gRNA RNA levels were calculated as a fold change relative to GAPDH using the 2-ACT method.
  • Nsp1-10 A 2NTD only DSJ levels were assessed due to absence of an USJ. The values corresponding to the USJ and DSJ were averaged to calculate DI levels.
  • DI levels were expressed relative to CoV-2 gRNA levels.
  • Fig. 9Q is a Western blot of 293 T/ACE2 cells stably expressing BirA (Ctrl, control) or Nsp1-10.
  • Fig. 10A is a graph showing a polysome analysis of 239T cells transfected with FLAG-NSp1 vector (30 pg). Cytoplasmic extracts were prepared 24 h post-transfections and polysomes analyzed by sucrose gradient sedimentation.
  • Fig. 10B is a graph showing a polysome analysis of 239T cells transfected with FLAG-NSp1 vector (KH/AA) (30 pg). Cytoplasmic extracts were prepared 24 h posttransfections and polysomes analyzed by sucrose gradient sedimentation.
  • Fig. 10C is a Western blot analysis of protein samples obtained from individual fractions in Fig. 10A. Fraction numbers are indicated to the top.
  • Fig. 10D is a Western blot analysis of protein samples obtained from individual fractions in Fig. 10B. Fraction numbers are indicated to the top.
  • Fig. 10E is a Coomassie stain of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified recombinant protein used in in vitro translation assays.
  • Fig. 10F is a graph showing in vitro inhibition of translation by recombinant Nsp1 , but not Nsp1-10.
  • the top panel shows a schematic diagram of FF/HCV/Ren mRNA reporter.
  • the figure shows the relative luciferase values obtained following in vitro translations of RRL programmed with 10 g/ml FF/HCV/Ren mRNA and supplemented with the indicated amounts of recombinant protein.
  • Fig. 10H is a Western blot of extracts prepared from the indicated cells demonstrating the expression of recombinant Nsp1-10 in 293T/ACE2 cells.
  • Fig. 101 is a graph demonstrating the reduction in virus titers obtained following infection of 293T/ACE2(Nsp1-10) cells, relative to titers obtained in 293T/ACE2 cells.
  • Fig. 11A is a shematic showing cohorts of C57BL/6 hACE2 mice intranasally infected with the indicated doses of stock, P1 , or P30 virus.
  • Fig. 11 B is a graph showing the change in body weight for the mice of Fig. 10A.
  • the dotted line represents the cutoff criteria for euthanasia.
  • Fig. 11C is a graph showing the change in clinical status for the mice of Fig. 11 A.
  • Grade 1 is pilorection and/or mild ruffled fur
  • grade 2 is mild hunched posture or mild ruffled fur and reduced activity
  • grade 3 is hunched posture and mild ruffled fur and reduced activity
  • grade 4 is hunched posture and ruffled fur and inactive;.
  • Fig. 11 D is a Kaplan-Meier curve showing survival of mice receiving the indicated viral doses and combinations.
  • Fig. 12A is a schematic depiction of the location of the Nsp12(A19) deletion in the RNA-bound RNA-dependent RNA polymerase (RdRp) complex (PDB 7BV2).
  • the deletion [575-594; shown in black] extends from the end of the fingers region (pink) to the beginning of the palm domain (green) and is predicted to impair binding to RNA.
  • Fig. 12B is a gel showing wild-type (WT) RdRp complexes (Nsp7, Nsp8, and Nsp12) or mutant complexes containing Nsp12 (SNN, i.e.
  • Nsp12 (A19) were expressed and purified from Baculovirus infected Sf-9 cells. These were used to assemble in vitro RNA synthesis reactions. Briefly, a 4-mer primer (5’pACGC3’) was extended with 0.1 M ATP, CTP, UTP, in the presence of 0.1 M [a- 32 P]GTP. Reaction products obtained in the presence or absence of WT RdRp complex were resolved on a denaturing 20% polyacrylamide gel. The Nsp12 (SNN) and Nsp12 (A19)RdRp complexes were significantly impaired for RNA synthesis (compare lanes 13-18 and 19-24 to lane 1 ).
  • polypeptides based on defective SARS-CoV-2 genomes are capable of squelching parental virus replication in vitro and in vivo.
  • nucleic acid molecules encoding defective viral genomes (DVGs) that are unable to replicate and/or be packaged on their own, but which still comprise replication and packaging signals, can function as defective interfering particles (DIPs).
  • DIPs compete with parental genomes for limited resources during replication and packaging, thereby leading to reduced parental virus titers.
  • polypeptides based on SARS-CoV-2 defective genomes or nucleic acid molecules encoding such a polypeptide are potential anti-coronavirus biologic drugs.
  • SARS-CoV-2 DVGs have been identified, and genetically and functionally characterized. All of the abundant SARS-Cov-2 DVGs isolated had: (i) retained 5’ end sequences, (ii) an in-frame fusion between Nsp1 and Nsp10 (the “Nsp1-10 fusion”), (iii) had retained a frameshift site and the Nsp12 ORF, and (iv) retained 3’ end sequences. It was demonstrated herein that the most abundant DVGs isolated can function as DI particles, genetically interfering with parental SARS-CoV-2 virus replication in vitro and in vivo.
  • the Nsp1-10 fusion product lacks the Nsp1 C-terminal sequences critical for interacting with the mRNA entry channel of the 40s ribosome and inhibiting host translation initiation during viral infection. Therefore, recombinant Nsp1-10 fusion proteins was constructed and showed that, unlike recombinant Nsp1 , the recombinant Nsp1-10 fusion protein was incapable of inhibiting cap-dependent translation when transfected into cells. It was further demonstrated that, upon infection with SARS-CoV-2, cells stably expressing Nsp1-10 exhibited viral yields that were reduced 25- fold compared to controls.
  • the present disclosure therefore relates to polypeptides capable of limiting the replication of coronavirus and/ or nucleic acid molecules encoding polypeptides capable of limiting the replication of coronavirus, including those comprising a Nsp1-10 fusion, and can provide an effective therapeutic treatment for coronaviruses.
  • DIP defective interfering particle
  • DVG defective viral genome
  • IFN interferon
  • MOI multiplicity of infection
  • NSP non-structural protein
  • ORF open reading frame
  • TIP therapeutic interfering particle
  • the present application provides an isolated polypeptide capable of limiting the replication of a coronavirus.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus.
  • the isolated polypeptide comprises a fusion of two polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11.
  • the isolated polypeptide comprises a fusion of three polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of four polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11.
  • the isolated polypeptide comprises a fusion of five polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of six polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11.
  • the isolated polypeptide comprises a fusion of seven polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of eight polypeptides encoded by Nsp1, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11.
  • the isolated polypeptide comprises a fusion of nine polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of ten polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11.
  • the isolated polypeptide comprises a fusion of 11 polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of ORF1a of a coronavirus and further comprises at least one polypeptide encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least two polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least three polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least four polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least five polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises the Nsp12 polypeptide, which includes the -1 ribosomal frameshift site.
  • ORFs viral open reading frames
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one coronavirus structural protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprise at least one of the S protein, E protein, M protein and N protein.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises each of the S protein, E protein, M protein and/or N protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one coronavirus accessory protein.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further at least one of the accessory proteins 3a, 6, 7ab and 8. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further at least two of the accessory proteins 3a, 6, 7ab and 8.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further at least three of the accessory proteins 3a, 6, 7ab and 8. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further each of the accessory proteins 3a, 6, 7ab and 8.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b and at least one structural protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b and at least one accessory protein.
  • the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b, at least one structural protein and at least one accessory protein.
  • ORFs viral open reading frames
  • the terms “fusion” or “fusion polypeptide” as used herein refer to a polypeptide created by the joining of two or more distinct polypeptides or proteins encoded by separate genes. The fusion polypeptide may be generated recombinantly through the genetic engineering of two or more separate ORFs of a ORF1a of a coronavirus into a single fusion gene.
  • the recombinant fusion polypeptide may further comprise linker genes to provide spacing between the different polypeptide components.
  • the fusion polypeptide may also be generated by linking two or more separate polypeptides or proteins together covalently, whether by peptide bonds or other types of chemical linkage (e.g. disulfide bonds) known in the art.
  • the fusion polypeptide may also be generated recombinantly from a naturally-occurring fusion gene.
  • replication refers to the action or process of reproducing or duplicating viral RNA and the associated viral proteins by the host cell and includes within this definition the assembly of infective progeny virus particles.
  • a polypeptide or nucleic acid is capable of “limiting the replication of a virus” when it reduces the amount of viral RNA and/or associated viral proteins by the host cell by at least half a log compared to a control.
  • the control may comprise an untreated host cell infected with parental virus.
  • the control may also comprise a host cell that is infected with parental virus and treated with a polypeptide encoding a reporter gene or a nucleic acid molecule encoding a polypeptide encoding a reporter gene, wherein expression of the reporter gene has not effect on the replication of the parental virus (e.g. luciferase).
  • Viral replication can be quantified using different standard techniques known in the art (e.g. plaque assay, quantitative real time polymerase chain reaction (qRT-PCR)).
  • At least two polypeptides fused comprise the coronavirus proteins Nsp1 (variants or fragments thereof) and Nsp10 (variants or fragments thereof), and the fusion polypeptide comprises a Nsp1 moiety and a Nsp10 moiety.
  • the term “moiety” as used herein refers to either a fragment of a polypeptide or the entire polypeptide.
  • a “variant” of a protein/polypeptide refers to a full length version of the protein/polypeptide, from any coronavirus, that comprises at least one amino acid difference when compared to the version of said protein/polypeptide normally found in the wild-type coronavirus.
  • fragment of a protein/polypeptide, as used herein, refers to a protein/polypeptide derived from, but shorter in length than, a full-length protein/polypeptide. The fragment retains the function of the peptide it is a fragment of.
  • the Nsp1 and Nsp10 polypeptides may be derived from wild-type SARS-CoV-2 Nsp1 and Nsp10 genes, respectively, or from variants of said genes, whether naturally occurring or engineered.
  • the isolated polypeptide may also comprise a fusion of Nsp1 and/or Nsp10 moieties derived from other coronaviruses including non-SARS-CoV-2 beta coronaviruses (e.g.
  • the isolated polypeptide comprises a Nsp1 moiety that comprises amino acid residues 1 to 162 of the SARS-Cov-2 wild-type Nsp1 (such as, for example, residues 1 to 162 of SEQ ID NO: 1 ).
  • the Nsp1 moiety can comprise the amino acid sequence of SEQ ID NO: 3, be a variant of the amino acid sequence of SEQ ID NO: 3 or be a fragment of the amino acid sequence of SEQ ID NO: 3.
  • the Nsp1 moiety can be a variant of the amino acid sequence of SEQ ID NO: 1 or a fragment of the amino acid sequence of SEQ ID NO: 1.
  • Variants of the Nsp1 moiety have, in some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1 or 3 and exhibits similar biological activity when compared to the wild-type amino acid sequence of SEQ ID NO: 1 or 3 .
  • the term “percent (%) identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences.
  • the level of identity between two or more sequences can be determined conventionally using known bioinformatics programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991 ).
  • the isolated polypeptide comprises a Nsp10 moiety that comprises amino acid residues 97 to 139 of the SARS-CoV-2 wild-type Nsp10 polypeptide (such as, for example, residues 97 to 139 of SEQ ID NO:2).
  • the Nsp10 moiety comprises the amino acid sequence of SEQ ID NO:4, be a variant of the amino acid sequence of SEQ ID NO:4, or be a fragment of the amino acid sequence of SEQ ID NO: 4.
  • the Nsp10 moiety can be a variant of the amino acid sequence of SEQ ID NO:2 or a fragment of the amino acid sequence of SEQ ID NO: 2.
  • Variants of the Nsp10 moiety have, in some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID 2 or 4 and exhibits similar biological activity when compared to the wild-type amino acid sequence of SEQ ID NO: 1 or 3.
  • the isolated polypeptide comprises a fusion of Nsp1i-162 (such as, for example residues 1 - 162 of SEQ ID NO: 1 or SEQ ID NO:3) and Nsp10 97 -i39 (such as, for example residues 97 - 139 of SEQ ID NO: 2 or SEQ ID NO:4) from SARS-CoV-2.
  • the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5, be a variant of the amino acid sequence of SEQ ID NO:5, or be a fragment of the amino acid sequence of SEQ ID NO:5.
  • Variants of the fusion of Nsp1i-i62 and Nsp10 9 7-i39 have, in some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 , 2, 3, 4 and/or 5 and exhibits similar biological activity when compared to the wild-type amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, and/or 5 (Table 1 ).
  • the present application also relates to an isolated nucleic acid molecule coding for the isolated polypeptide described herein.
  • this nucleic acid molecule comprises sequential sequences encoding for the at least two polypeptides from the ORF1a of a coronavirus that are operably linked in the same open reading frame and is capable of expressing the isolated polypeptide described herein.
  • the isolated nucleic acid molecule comprises one or more ribonucleic acid (RNA) residues.
  • the isolated nucleic acid molecule is a messenger RNA molecule (mRNA).
  • mRNA messenger RNA molecule
  • the isolated nucleic acid molecule comprises one or more deoxyribonucleic acid (DNA) residues.
  • the isolated nucleic acid molecule is a DNA molecule.
  • the isolated nucleic acid molecule further comprises (a) heterologous gene(s).
  • suitable heterologous genes include genes that can help boost and antiviral response such as “kill-switches”, interferons (IFNs), short hairpin RNA (shRNA) and/or Cas9.
  • An inducible promoter may be used to drive expression of the heterologous gene.
  • the present disclosure provides an isolated nucleic acid molecule comprising a defective viral genome (DVG) encoding a therapeutic interfering particle (TIP).
  • DVG defective viral genome
  • TIP therapeutic interfering particle
  • a “DVG” comprises a coronavirus genome that, due to the emergence of one or more mutation, is lacking essential cis-acting elements and/or is unable to replicate on its own.
  • the DVGs result from errors during replication that led to complementary ends, deleterious point mutations, deletions, insertions, mosaic rearrangements, or any combination of these.
  • coronavirus DVGs may be obtained by growing the virus in host cells under appropriate conditions (e.g. a high multiplicity of infection for 15 - 30 passages).
  • a “TIP” comprises a DVG that has lost a critical portion normally required for the ability of the virus to replicate and/or be packaged, but has retained replication and packaging signals that allow it to be replicated and be packaged in the presence of a complete functional virus genome capable of providing any missing functions.
  • the TIP is capable of limiting the replication of a human coronavirus.
  • the ability of the TIP to limit replication of a human coronavirus can be determined by comparing the viral titers obtained from host cells infected with a human coronavirus and the TIP, compared to control cells that are only infected with a human coronavirus. Viral titers can be measured using standard techniques known in the art.
  • the TIP is replication defective and can be replicated in the presence of a helper virus.
  • helper virus refer to a virus that allows an otherwise-deficient co-infecting virus to replicate, by providing the functions that they have lost. Standard techniques known in the art can be used to assess whether or not a TIP is replication defective on its own in host cells and whether it can become replication competent in the presence of a helper virus.
  • the TIP is defective for packaging and can be packaged in the presence of the helper virus. Standard techniques known in the art can be used to assess whether or not a TIP is defective in packaging on its own in host cells and whether it can become capable of being packaged in the presence of a helper virus.
  • the TIP is capable of being enriched upon a plurality of passages in a cell infected with the coronavirus and/or the helper virus at a multiplicity of infection equal to or greater than 1 .
  • the terms “passage,” “passaging” or “serial passaging” refers to the in vitro or in vivo process of growing a coronavirus in iterations whereby the virus is allowed to grow for a certain amount of time in a given environment before part of it will be transferred to a new environment and allowed to grow for the same period. This process will be repeated as many times as desired.
  • Enrichment of the TIP following a plurality of passages in a cell infected with coronavirus and/or the helper virus at a multiplicity of infection equal to or greater than 1 PFU per cell can be determined using standard techniques known in the art such as, for example, qRT-PCR.
  • the TIP encodes the isolated polypeptide defined herein and/or comprises the isolated nucleic acid molecule defined herein.
  • the human coronavirus is the helper virus that provides the functions needed to allow for the TIP to replicate and/or be packaged.
  • the human coronavirus and/or the helper virus is from the alpha coronavirus genus.
  • the human coronavirus and/or the helper virus are 229E or NL63. In other embodiments, the human coronavirus and/or helper virus is from the beta coronavirus genus. In yet other embodiments, the coronavirus and/or helper are 0043, HKU1 , SARS-CoV, MERS-CoV, or SARS-CoV2. In other embodiments, the human coronavirus and/or helper virus is from the gamma coronavirus genus. In yet other embodiments, the human coronavirus and/or helper virus is from the delta coronavirus genus.
  • the isolated nucleic acid molecule that comprises the DVG encoding a TIP comprises, when compared to the nucleic acid sequence of the genome of the coronavirus and/or the helper virus, a first deletion in a ORF1a.
  • the first deletion starts at a position corresponding to position 749 of GeneBank accession number NC_045512.
  • the first deletion ends at a first end position corresponding to position 13311 of GenBank accession number NC_044512.
  • the first deletion encompasses the nucleic acid sequence encoding at least one of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9.
  • the first deletion encompasses Nsp2. In some embodiments, the first deletion encompasses Nsp3. In some embodiments, the first deletion encompasses Nsp4. In some embodiments, the first deletion encompasses Nsp5. In some embodiments, the first deletion encompasses Nsp6. In some embodiments, the first deletion encompasses Nsp7. In some embodiments, the first deletion encompasses Nsp8. In some embodiments, the first deletion encompasses Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least two of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9.
  • the first deletion encompasses Nsp2 and Nsp3. In some embodiments, the first deletion encompasses Nsp3 and Nsp4. In some embodiments, the first deletion encompasses Nsp4 and Nsp5. In some embodiments, the first deletion encompasses Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp6 and Nsp7. In some embodiments, the first deletion encompasses Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp8 and Nsp 9.
  • the first deletion encompasses the nucleic acid sequence of at least three of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3 and Nsp4. In some embodiment, the first deletion encompasses Nsp3, Nsp4 and Nsp5. In some embodiments, the first deletion encompasses Nsp4, Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp5, Nsp6 and Nsp7. In some embodiments, the first deletion encompasses Nsp6, Nsp7 and Nsp8.
  • the first deletion encompasses Nsp7, Nsp8 and Nsp9 In some embodiments, the first deletion encompasses the nucleic acid sequence of at least four of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3, Nsp4 and Nsp5. In some embodiments, the first deletion encompasses Nsp3, Nsp4, Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp4, Nsp5, Nsp6 and Nsp7.
  • the first deletion encompasses Nsp5, Nsp6, Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp6, Nsp7, Nsp8 and Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least five of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3, Nsp4, Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp3, Nsp4, Nsp5, Nsp6 and Nsp7.
  • the first deletion encompasses Nsp4, Nsp5, Nsp6, Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp5, Nsp6, Nsp7, Nsp8 and Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least six of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3, Nsp4, Nsp5, Nsp6 and Nsp7.
  • the first deletion encompasses Nsp3, Nsp4, Nsp5, Nsp6, Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 and Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least seven of Nsp2, Nsp3, Nsp4,
  • the first deletion encompasses the nucleic acid sequence of each of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 and Nsp9.
  • the TIP described herein must be capable of limiting replication of a human coronavirus.
  • the TIP is capable of limiting the titers of a human coronavirus and/or helper virus by at least one log or more.
  • the “titer” of a virus refers to the concentration of infectious viral particles. Infectious viral particle titers may be quantified using techniques known in the art such as, for example, plaque formation assays, focus formation assays, end point dilution assays and protein- based virus quantification assays.
  • the DVG encoding a TIP has the nucleic sequence of GI.285 (SEQ ID NO:6), GI.249 (SEQ ID NO:7), GI.616 (SEQ ID NO:8), GI.50 (SEQ ID NO:9), GI.55 (SEQ ID NO:10) or GI. 535 (SEQ ID NO:11 ) or a corresponding mRNA version of these nucleic acid sequences (Table 2).
  • DVGs with nucleic sequences that comprise intact replication and packaging sequences, such as the aforementioned DVG sequences, are capable of replicating and expanding in the presence of wild-type and/or helper viruses.
  • the DVGs can include additional heterologous genes, such as, for example, “kill-switches”, interferons (IFNs), inducible Caspase triggers, short hairpin RNA (shRNA) and/or Cas9.
  • the DVGs can be delivered as a therapeutic interfering particle.
  • the DVGs can be delivered as a nucleic acid vector to be expressed in coronavirus-infected cells. Such DVGs may be prepared in vitro by genetic engineering using standard techniques and delivered as a therapeutic. The nucleic sequence of the DVG can be determined using standard sequencing techniques known in the art.
  • the present disclosure provides a TIP comprising the isolated poypeptide described herein and/or the isolated nucleic acid molecule described herein.
  • the TIP comprising the isolated polypeptide and/or the isolated nucleic acid may be generated by repeated serial passaging of a parental coronavirus, at high multiplicities of infection (MOI) in a suitable cell line.
  • MOI multiplicities of infection
  • a high MOI, for the purpose of generating DVGs and TIPs, is known in the art and includes, but is not limited to, MOIs of 1 - 10 particle forming units (PFU) per cell.
  • the TIP comprising the isolated polypeptide and/or the isolated nucleic acid may be generated by co-transfecting cells with a synthetic recombinant DVG genome and the genome of a helper virus. Similarly, cells lines that stably express synthetic recombinant DVG genome and the genome of a helper virus can be used to generate the TIP comprising the isolated polypeptide and/or the isolated nucleic acid.
  • the synthetic recombinant DVG genome may be prepared using standard techniques known in the art.
  • the TIP comprising the isolated isolated polypeptide and/or the isolated nucleic acid may comprise an engineered virus-like particle (VLP) that encapsulates the isolated nucleic acid and iscapable of delivering it into a recipient cell infected with coronavirus. Techniques for the preparation of VLPs and the delivery of proteins to cells via VLPs are known in the art.
  • VLP engineered virus-like particle
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (i) the isolated polypeptide derived herein and/or the isolated nucleic acid defined herein, and (ii) one or more pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient is known in the art and includes, but is not limited to, 0.01 - 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable excipients may be aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • the pharmaceutical composition is formulated for nasal administration.
  • the present disclosure provides a method of treating a coronavirus infection in a subject in need thereof, whereby the method comprises administering a therapeutically effective amount of the isolated polypeptides defined herein, the isolated nucleic acid defined herein and/or the pharmaceutical composition defined herein to the subject so as to reduce the replication of the coronavirus.
  • therapeutically effective amount refers to a quantity of the isolated polypeptide described herein, the isolated nucleic acid described herein and/or the pharmaceutical composition defined herein that is effective in mitigating at reducing replication of the coronavirus and/or mitigating one or more symptom of coronavirus infection when administered to an individual in need thereof. It is also understood herein that a therapeutically effective amount of the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition may be provided in different dosage forms and by different routes, both alone or in combination with other therapeutic agents indicated for the treatment of human coronavirus infections (e.g. remdesivir, sotrovimab, dexamethasone).
  • remdesivir sotrovimab, dexamethasone
  • the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided intra-nasally to the subject in need thereof. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided by inhalation. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided by injection. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided orally. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to the subject in need thereof in one or more doses.
  • the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof after the presence of a coronavirus is detected in the subject. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof before the presence of a coronavirus is detected in the subject. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof before and after the presence of a coronavirus has been detected in the subject.
  • the method of treating a coronavirus infection in a subject in need thereof comprises prophylactically administering a therapeutically effective amount of the isolated polypeptides defined herein.
  • prophylactically administering refers to administering a therapeutically effective amount of the isolated polypeptide, the isolated nucleic acid and/or the pharmaceutical composition defined herein to a subject before they are exposed to and/or infected by coronavirus, including but not limited to before the presence of coronavirus is detected in the subject.
  • prophylactically administering can also refer to administering a therapeutically effective amount of the isolated polypeptide, the isolated nucleic acid and/or the pharmaceutical composition defined herein to a subject after exposure to and/or infection by coronavirus, including but not limited to after the presence of coronavirus has been detected in the subject, but before the development of symptoms of coronavirus infection.
  • the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof prophy lactically and in combination with other prophylactic agents.
  • the subject in need thereof is a mammal.
  • the subject in need thereof is a human being.
  • the subject in need thereof is a child.
  • the subject in need thereof is an adult.
  • the present disclosure thus provides polypeptides capable of limiting the replication of coronavirus and/ or nucleic acid molecules encoding polypeptides capable of limiting the replication of coronavirus, including those comprising a Nsp1-10 fusion and/or Nsp12.
  • Virus and cells Vero E6, HEK 293T, and HEK 293T-ACE2 (PMID 34110264) cells obtained from the American Type Culture Collection (Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 100 U/ml penicillin and streptomycin, 1 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (Gibco), 2 mM glutamine (Gibco), and 10% heat-inactivated fetal bovine serum (FBS) at 37°C in 5% CO 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
  • FBS heat-inactivated fetal bovine serum
  • SARS-CoV-2 (hCoV-19/Canada/CN-VIDC-01/2020; GISAID accession no. EPI_ISL_425177) was a donation.
  • HEK 293T-ACE2 cells stably expressing Nsp1-10 HEK 293T-ACE2 cells were transfected by calcium phosphate with pcDNA3.1- 3xFLAG-Nsp1/10 (5 pg). Expression of Nsp1-10 was verified by Western blotting and probing with a monoclonal anti-FLAG antibody (1 :1000 dilution) produced in mouse (Sigma- Aldrich, #F1804).
  • Vero E6 cells 106 cells/well grown in six-well plates were infected with SARS-CoV-2 at MOI of 3 for 60 min. The virus was removed and replaced with 2 mL of fresh complete DMEM media. Cells were monitored daily, and viral supernatant was harvested after the appearance of cytopathogenic effect (CPE) between 24 and 48 h. Fifty percent of the supernatant containing passage 1 (P1 ) of the virus was used to infect a new batch of Vero E6 cells, and the remaining R1 virus was frozen as two equal-volume aliquots at -80 °C. After 1 h of infection, the virus was removed and replaced with 2 ml_ fresh complete DMEM media.
  • CPE cytopathogenic effect
  • Antibodies used in the examples were: anti-Nsp1 (GeneTex, GTX135612), anti-Nsp10 (Pro-Sci Inc, #9179), anti-RPS6 (CST, #2317 ), anti-FLAG (Sigma- Aldrich, #F1804), anti-RPL7 (Novus Biologicals, #NB100-2268, anti-GAPDH (Abeam, #ab8245), anti- -actin (Abeam, #ab8226), anti-eEF2 (CST, #2332), and antihnRNPAI (CST, #8443).
  • pHiC IntIK G1.616 To generate pHiC IntIK G1.616, the EMCV-renilla luciferase sequence released from pHiC IntU G1.616 by restriction enzyme digestion using Alel and Stul and was replaced with a G block non-directionally containing Nsp13-N sequence bounded by Alel and Stul restriction sites. pHiC IntI K G1.50 was generated in the same manner as pHiC IntI K G1.616, but starting with the pHiC IntU G1.50 vector, instead of pHiC IntU G1.616. pHiC IntI K G1.55 was cloned in two steps.
  • a G block consisting of the SARS-CoV-2 5’ UTR, Nsp1-10 fusion protein, Nsp11, and Nsp12 N- terminus bounded by Sphl and Sacl restriction sites was directionally cloned into pHiC IntU G1.50 to give pHiC IntU G1.55.
  • the EMCV-renilla was released from pHiC IntU G1.55 by restriction enzyme digestion using Sacl and EcoRI and a G block that has Sacl and EcoRI restriction sites and contains the Nsp12 C-terminus, Nsp13-N fusion sequence, and ORF10 (aa 1-21 ) was then cloned directionally into pHiC IntU G1.55.
  • pcDNA3.1_Nsp1-3xFLAG containing a Nhel site downstream of the CMV promoter and a BamHI site upstream of the BGH pA signal was a donation.
  • the Nsp1-3xFLAG insert was removed by restriction digest with Nhel/BamHI.
  • a G block containing the Nhel/BamHI restriction sites and 3xFLAG-spacer-Nsp1 sequences was directionally cloned to generate pcDNA3.1_3xFLAG-Nsp1.
  • pcDNA3.1_3xFLAG-Nsp1 K164A/H165A
  • a two-step cloning was implemented: In the first step, a PCR product was amplified from cDNA obtained from Exp#1 P30 as template using the primers Narl-f (5’AGTCCACAAGCACGGCGCCGATCTAAAGTCA3’ SEQ ID NO: 12) and Narl/BamHI-r
  • Narl/BamHI restriction digest of pcDNA-3.1_3xFLAG-Nsp1 released three fragments. After cutting with Narl, the PGR product was cloned into the linearized backbone and appropriate recombinants selected. In the second step, the BamHI/Narl fragment from pcDNA-3.1_3xFLAG-Nsp1 was directionally sub-cloned into one of the selected recombinants.
  • pET15b-His6-Nsp1 pET15b-His6-Nsp1/10, and pET15b-His6-Nsp1 (K164A/H165A) were cloned using G blocks with Ndel and BamHI positioned at the ends. All cloned G blocks were Sanger sequenced to ensure the absence of mutations.
  • Uncapped mRNA was capped in a one-step reaction using Vaccinia Capping System (NEB, # M2080S) and mRNA Cap 2’-O-Methyltransferase (NEB, #M0366S).
  • RNA clean-up was performed via phenol-chloroform extraction as described above for clean-up of linearized DNA. Concentrations were quantitatively measured by NanoDropTM 1000 (Thermo Scientific).
  • Cap-1 mRNA was analyzed alongside ssRNA ladder (NEB, # N0362S) on a 1% agarose-formaldehyde denaturing gel to confirm size and quality.
  • RNA transfections VeroE6 cells were seeded in a 24-well plate in DMEM supplemented with 10% FBS (Gibco, #12483-020, 1% penicillin-streptomycin (Wisent, # 450- 200-EL), and 1x non-essential amino acids (Wisent, # 321-011-EL) at a density of 2x10 5 cells/well. The next day, the medium was changed to 200 pL opti-MEM (Gibco, 31985070). Cells were transfected with LipofectamineTM 3000 (Invitrogen, #L3000015), and transfection mixes were prepared as recommended by the manufacturer. Essentially, 500 ng of cap-1 mRNA was added to each well of mock- or SARS-CoV-2 infected cells and left to incubate for 22 hours before downstream processing.
  • FBS Gibco, #12483-020, 1% penicillin-streptomycin (Wisent, # 450- 200-EL), and 1x non-essen
  • RNA extraction Total RNA was extracted from viral supernatants using QIAampTM Viral RNA Mini Kit according to the manufacturer’s instructions (Qiagen, # 52904). Total RNA was extracted from SARS-CoV-2 infected cells using the Nucleospin RNA mini kit for RNA purification according to the manufacturer’s instructions (Macherey-Nagel, #740955.50). For isolation of RNA from DI RNA-transfected, uninfected cells, cells were lysed with 500 pL TRIzolTM directly in the plates and incubated for 5 minutes. Total RNA was extracted according to the manufacturer’s instructions (Invitrogen).
  • RNA ladder lane was excised and stained with SYBRTM Gold Nucleic Acid Gel Stain (Invitrogen). Northern blot transfers were performed onto Hybond N+ membrane as previously described using 20 x SSC.
  • the membrane was UV-crosslinked at 1.2 x 10 5 uJ/cm 2
  • the membrane was pre-hybridized with hybridization buffer (50% formamide, 10% dextran sulfate, 0.8M NaCI, 5x Denhardt’s solution, 50mM Tris 7.5, 0.1% sodium pyrophosphate, 100ug/mL salmon sperm DNA, 0.5% SDS) for 16 hours at 42°C, hybridized with the radioactively probe for 16 hours at 42°C. Washes were performed at 65°C twice for 25 minutes each with 0.1% SDS/2x SSC, 0.1% SDS/1x SSC, and 0.1% SDS/0.5x SSC. Autoradiographs were obtained by exposing the membrane to X-ray film (BioMaxTM XAR, Kodak).
  • RNA nanopore sequencing Before sequencing, the extracted total RNA was quantified with the “Qubit RNA high sensitivity” quantification kit (Q32855 ; ThermoFisher Scientific) and its quality was profiled on a “High Sensitivity RNA ScreenTape” (5067-5579 ; Agilent). Only high-quality samples were sequenced. The total RNA was sequenced on a MinlON flow-cell (FLO-MIN106 ; Oxford Nanopore Technologies) using the “Direct RNA sequencing” library preparation kit (SQK-RNA002 ; Oxford Nanopore Technologies).
  • FLO-MIN106 MinlON flow-cell
  • SQK-RNA002 Oxford Nanopore Technologies
  • the SQK-RNA002 library preparation protocol was followed (version DRS_9080_v2_revM_14Aug2019) as provided from Oxford Nanopore Technologies (abbreviated as ONT) with the following modifications.
  • the library preparation started with 1 pg of total RNA for the passages 1 and 29 of experiment number #1, 3 pg of total RNA for the passage 15 of experiment number #1 and 2 pg of total RNA for the passages 1 , 14 and 30 of experiment number #2. In cases where the starting material was 1 pg of total RNA, the following protocol was used.
  • the first adaptor of the library preparation kit was ligated on the RNA in a 15 pl solution with the following components: 3 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 1 pg of total RNA, 0.5 pL of Recombinant RNase Inhibitor (stock: 40 Units/ul ; 2313A ; Takara), 1 pL of RT Adapter (RTA ; ONT); 1.5 pL of T4 DNA ligase (stock: 2M U/ml ; M0202 ; New England Biolabs), top up the solution to 15 pL with nuclease-free water.
  • This solution was incubated at room temperature for 20 minutes and subsequently mixed with a 23 pL solution named “reverse transcription master mix” that had the following components: 9 pL of nuclease-free water, 2 pL of 10mM dNTPs (N0447S ; New England Biolabs), 8 pL of 5x SuperscriptTM IV reverse transcription (SSIV RT) buffer (18090010 ; ThermoFisher Scientific), 4 pL of 0.1 M dithiothreitol (DTT) (18090010 ; ThermoFisher Scientific).
  • RNACIean XP beads cleanup
  • the material was then eluted from the beads with 20 pL of nuclease-free water and the second adaptor was ligated in a 40 pL solution containing the following: 20 pL of reverse-transcribed RNA, 8 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 6 pL of RNA Adapter (RMX ; ONT), 2.5 pL of nuclease-free water, 0.5 pL of Recombinant RNase Inhibitor (stock: 40 Units/ul ; 2313A ; Takara), 3 pL of T4 DNA Ligase (stock: 2M U/ml ; M0202 ; New England Biolabs).
  • RNACIean XP “RNACIean XP” beads cleanup (A63978 ; Beckman Coulter) and two washes of 150 pL with the Wash Buffer (WSB; ONT).
  • the material was eluted, from the beads, in 21 pL of Elution Buffer and 1 pL of the solution was quantified with the “Qubit 1X dsDNA high sensitivity” kit (Q33230 ; ThermoFisher Scientific). Approximately 200-250 ngs of RNA/cDNA hybrid were recovered.
  • RNA/cDNA hybrid 20 pL of prepped RNA/cDNA hybrid in Elution Buffer, 17.5 pL of nuclease-free water, 37.5 pL of Rapid Running buffer (RRB) (ONT).
  • RRB Rapid Running buffer
  • the duration of the sequencing run was up to 72 hours or until no pores were available for sequencing. In cases where the starting material was 2 or 3 pg of total RNA, the following protocol was used.
  • the first adaptor of the library preparation kit was ligated on the RNA in a 30 pL solution with the following components: 6 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 2 or 3 pg of total RNA, 1 pL of Recombinant RNase Inhibitor (stock: 40 Units/pL ; 2313A ; Takara), 1 pL of RT Adapter (RTA ; ONT); 3 pL of T4 DNA ligase (stock: 2M U/ml ; M0202 ; New England Biolabs), top up the solution to 30 pL with nuclease-free water.
  • the solution was incubated at room temperature for 20 minutes and subsequently mixed with a 46 pL solution named “reverse transcription master mix” with the following components: 18 pL of nuclease-free water, 4 pL of 10mM dNTPs (N0447S ; New England Biolabs), 16 pL of 5x SSIV RT buffer (18090010 ; ThermoFisher Scientific), 8 pL of 0.1 M DTT (18090010 ; ThermoFisher Scientific).
  • RNACIean XP beads cleanup
  • the material was then eluted from the beads with 40 pL of nuclease-free water and the second adaptor was ligated in a 80 pL solution containing the following: 40 pL of reverse-transcribed RNA, 16 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 6 pL of RNA Adapter (RMX ; ONT), 11 pL of nuclease-free water, 1 pL of Recombinant RNase Inhibitor (stock: 40 Units/pL ; 2313A ; Takara), 6 pL of T4 DNA Ligase (stock: 2M U/mL ; M0202 ; New England Biolabs).
  • RNACIean XP “RNACIean XP” beads cleanup (A63978 ; Beckman Coulter) and two washes of 150 pL with the Wash Buffer (WSB; ONT).
  • the material was eluted, from the beads, in 38.5 pL of Elution Buffer and 1 pL of the solution was quantified with the “Qubit 1X dsDNA high sensitivity” kit (Q33230 ; ThermoFisher Scientific). Approximately 400-750 ngs of RNA/cDNA hybrid were recovered.
  • RNA sequencing data processing RNA sequencing data for all passages were base called by guppy 3.4.4 (Oxford Nanopore Technologies) using the high-accuracy model. The sequenced reads were first adapter-trimmed using porechop (https://github.com/rrwick/Porechop) and then aligned to the reference sequence database consisted of the SARS-CoV-2 genome (GenBank: NC_045512.2), yeast ENO2 cDNA (SGD: YHR174W), human ribosomal DNA complete repeat unit (GenBank: U13369.1 ), C.
  • JUNCTIONS file was downloaded from UCSC Table browser (track ‘Transcriptome Kim’, table ‘Known transcripts’). Chimeric reads, secondary and supplementary alignments were filtered out according to the flags from minimap2 using samtools v1.11 (http://www.htslib.org/, PMID: 19505943) and picard V2.23.8 (https://github.com/broadinstitute/picard). Mapped transcripts were collapsed into transcript models using PINFISH (https://github.com/nanoporetech/pinfish.git), RATTLE
  • RT-qPCR Complementary DNA was generated either with M-MuLV reverse transcriptase (NEB, M0253L) or SuperscriptTM IV VILOTM Mastermix (ThermoFisher, #11756050) using random hexamer primers.
  • the cDNA was diluted 10-fold and used as template for qPCR using SsoFastTM EvagreenTM Supermix (Bio-Rad, #1725201 ). Cycling conditions consisted of an initial denaturation of 95°C/30 seconds followed by 98°C/5 seconds, 60°C/5 seconds (39 cycles), and 65°C to 95°C incremented at a rate of 0.5°C/min. for melt curve acquisition. Primer pairs used are listed in Table 4.
  • HEK293T cells were seeded in 10 cm dishes at 5x10 s cells/well in DMEM supplemented with 10% BGSS, 1% penicillin/streptomycin, and 1% L- glutamine (Wisent). The next day, cells were transfected by calcium phosphate using 10 pg of each plasmid. Cells were washed and fresh medium re-applied 6-8 hours posttransfection. Twenty-four hours post-transfection, cells were harvested in ice cold PBS containing 100 pg/ml cycloheximide.
  • the gradients were centrifuged at 217,290 xg for 2 h at 4°C in an SW40 Beckman rotor. Fractions were collected using the Teledyne ISCO Foxy R1 collector while monitoring the UV 254 profile. Proteins were precipitated from each fraction with 10% trichloroacetic acid and collected by centrifugation at 16,000 x g for 30 min 4°C. The pellet was washed with 500 pL acetone, centrifuged at 4°C for 10 min. at 16,000 x g, and dried under vacuum (Eppendorf Vacufuge). Protein pellets were resuspended in 1x SDS sample buffer and analyzed on a 10% SDS-PAGE gel. Resolved proteins were transferred at 4°C onto a polyvinylidene difluoride (PVDF) membrane (Bio-RadTM) and probed by immunoblotting.
  • PVDF polyvinylidene difluoride
  • HEK 293T cells were seeded in a 6-well plate at a density of 1x10 s cells/well in DMEM supplemented with 10% BGSS, 1% penicillin-streptomycin, and 1% L-glutamine.
  • Labelling was performed for 15 min at 37°C/5% CO2 after which cells were lysed in RIPA buffer (20mM Tris-CI 8.0, 100mM NaCI, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.01 mg/ml aprotinin, 0.002 mg/ml leupeptin, 2.5uM pepstatin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)).
  • RIPA buffer 20mM Tris-CI 8.0, 100mM NaCI, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'
  • TCA trichloroacetic acid
  • CPMs were normalized to the total protein amounts determined for each sample by the DC Protein Assay (Bio-Rad, #5000112).
  • Radiolabeled lysates were also resolved by SDS-PAGE and transferred onto a PVDF membrane (Bio-RadTM) when Western blots had to be performed.
  • Recombinant protein purification Recombinant His6-tagged Nsp1 , Nsp1 (K164A/H165A) and Nsp1/10 proteins were purified BL21 (DE3) cells expressing pET15b- based expression vectors. Single colonies were picked and 20 ml_ cultures were grown overnight in LB media supplemented with ampicillin (100ug/ml). Cultures were used to inoculate 1 L of LB/amp (100 pg/ml) and induced with isopropylthio-p-galactoside (IPTG) (0.5 mM) when the OD600 reached 0.8, at which point cultures were moved to 18°C for 16 hours.
  • IPTG isopropylthio-p-galactoside
  • Nsp1 sonication 50mM HEPES-KOH, 500mM KCI, 5mM MgCI 2 , 40mM imidazole, 10% glycerol, 1 mM PMSF, 0.01 mg/ml aprotinin, 0.002 mg/ml leupeptin, 2.5uM pepstatin, 0.5mM dithiothreitol, pH 7.6), and lysed by sonication(Heat systems ultrasonics; 10 pulses at 1 pulse/sec). The lysate was cleared by centrifugation at 4°C for 48 minutes at 48,000 x g.
  • Proteins were purified on Ni-NTA agarose beads (Qiagen, #30210), washed twice with Nsp1 sonication buffer, and eluted with Nsp1 elution buffer (50mM HEPES-KOH, 500mM KCI, 5mM MgCI 2 , 300mM imidazole, 10% glycerol, pH 7.6). Eluted protein fractions were dialyzed overnight at 4°C in Nsp1 dialysis buffer (40mM HEPES-KOH, 200mM KCI, 4mM MgCI 2 satisfy 10% glycerol, 1 mM dithiothreitol, pH 7.6). Protease inhibitors and dithiothreitol were always freshly added.
  • mice K18-ACE2-overexpressing mice (Stock No: 034860) were obtained from the Jackson Laboratory (Bar Harbor, Maine) and maintained by back- crossing to strain C57BL/6J (Stock No. 000664). Mice were infected with 10 4 PFU intranasally under ketamine-xylazine anaesthesia. Body weight and clinical score was monitored at least daily and twice at peak seakness days (days 5-7 post infection). Clinical signs were given a value 0 to 4 according to the scale: 0, normal appearance and behavior; 1 , hunched but active; 2, hunched and milde ruffled and less activity; 3, moderate hunched posture, ruffled fur, less active, shallow breathing. Mice were humanely sacrificed upon reaching 20% weight-loss or a clinical score of 3.
  • Plaque assays Vero E6 cells were plated in 24-well plates (105 cells/well) and incubated overnight at 37 °C. Virus-containing media was serially diluted (10“ 1 -10“ 6 ) with DMEM media into 96-well plates. Then, 100 L of each dilution was added in duplicate to Vero E6 cells in the 24-well plates, and samples were incubated at 37 °C in 5% CO 2 for 1 h with rocking every 15 min. After 1 h incubation, the virus-containing media was removed and 1 mL of pre-warmed plaquing media (MEM media containing 2% FBS and 0.75% methylcellulose) was added to each well.
  • MEM media pre-warmed plaquing media
  • renilla luciferase assay reagent Promega, #E2810
  • luciferase activity was measured immediately using a Synergy HTX plate reader (Biotek; Winooski, VT, USA).
  • the remaining virus was collected for titering.
  • total cellular RNA from each passage of cells was also extracted and stored at -80 °C for future use.
  • the major long-range product was gel purified and cloned by TA cloning into pGEM-T Easy using the pGEMTM-T Easy Vector System (Promega, # A1360) according to the manufacturer’s instructions or by blunt-end ligation into pBluescript II KS (+) using EcoRV. Minipreps were performed by the alkaline lysis method to obtain plasmid DNA from each clone which was then Sanger sequenced.
  • Vero E6 cells were infected with SARS-CoV-2 at an MOI of 1 , and DI mRNA was transfected 8 h post-infection. Twenty-two hours posttransfection, the resulting supernatant was serially passaged three times, once every 24 h. Cellular RNA was extracted at the indicated time points postinfection in P2 (4, 8, and 24 h) and at 4 h in P3. RNA was also extracted from P2 supernatant at 24 h. After performing RT- qPCR, the growth rate of SARS-CoV-2 was then calculated by the 2-ACt method using GAPDH as a control.
  • the resulting values were normalized to the 4 h time point.
  • the DI and SARS-CoV-2 RNA copy numbers were determined after RT-qPCR, from a standard curve established using recombinant RNA standards.
  • the percentage of packaged DI or WT virus mRNAs was then calculated as follows: 100 * (mRNA copy number in P2 supernatant)/(mRNA copy number in P2 supernatant + mRNA copy number in P2 cells).
  • the transmission percentage was calculated as follows: 100 * (mRNA copy number in P3 cells at 4 hr)/(mRNA copy number in P2 cells at 24 h).
  • HEK-293T cells were seeded in a six-well plate at a density of 10 6 cells/well. In each well, cells were transfected by calcium phosphate using 5 pg empty pcDNA3.1 or pcDNA3.1 expressing the indicated proteins. Twenty-four hours post-transfection, cells were scraped in cold PBS and pelleted by centrifugation at 4 °C for 10 min at 300 * g.
  • digitonin extraction buffer (10mM 1,4- Piperazinediethanesulfonic acid (PIPES) (pH 6.8), 300mM sucrose, 100mM NaCI, 3mM MgCI 2 , 5mM EDTA, 0.015% digitonin, 1 mM PMSF) on ice for 10 min, and the lysate was centrifuged at 4 °C for 10 min at 480 * g. The supernatant was kept as the cytosolic fraction. The digitonin-insoluble pellet was then washed once in the same volume of digitonin extraction buffer and spun at 480 * g for 10 min.
  • PPES 1,4- Piperazinediethanesulfonic acid
  • Triton-X-100 extraction buffer (10 mM PIPES (pH 6.8), 300mM sucrose, 100mM NaCI, 3mM MgCI2, 5mM EDTA, 0.5% Triton-X-100, 1 mM PMSF), left on ice for 15 min, and was centrifuged at 5000 * g for 10 min. at 4 °C. The supernatant (membrane/organelle fraction) was discarded, and the Triton-insoluble pellet was lysed in 100 pL of 1x sample buffer to obtain the nuclear fraction. The same cell equivalents of cytosolic and nuclear fractions were resolved on a 10% SDS-PAGE gel, and proteins were analyzed by western blotting.
  • DRS Direct RNA sequencing
  • the 5 kb DVGs were first detectable at P20 and persisted up to P30, indicating these were capable of replicating and being packaged in the presence of parental virus.
  • a large population of heterogeneous DVGs was detected by the 3’ end G probe in the latter passages (ie, P20-P30) as smears in the lanes that were absent in the RNA samples from mock-infected cells or that were less abundant in early passage virus stocks (P2-P16) (Figs. 1 F-1G).
  • Nanopore sequencing of RNA from P1 , P15 and P29 infected cells revealed a complex set of DVGs emerging at P15 and P29 (Fig. 1 H).
  • a -4.7 kb DVG retaining nucleotides 13,311/13,312-16,841 had emerged.
  • P30 GI.535 and two related genomes, GI.1634 and GI.1650 (differing only in the 5' end starting location), predominated the DVG population (Fig. 10).
  • GI.535 harbors: (i) an Nsp1-10 in-frame fusion, (ii) sequences spanning Nsp11 , the frameshift signal, and Nsp12, and (iii) an out-of-frame fusion between Nsp13 and the last 116 nts of the N ORF (Fig. 1 P).
  • GI.535 was the dominant DVG (-83%) throughout P20-P30, indicating stable long-term propagation of this genome (Figs. 1T-1V).
  • Nanopore sequencing enabled facile identification and abundance determination of DVG transcript models harboring multiple deletions during SARS-CoV-2 passaging.
  • the high error rate (-10%) associated with this technology made it imperative that the sequences of the more abundant DVGs of interest be verified by Sanger sequencing. This was achieved by undertaking long range PGR to amplify the abundant DVGs identified at P14 (Figs. 2A-2D) and P30 (Figs. 2E-2H) using primers anchored to the 5’ and 3’ ends of the SARS-CoV-2 genome (Primers A1/A2).
  • primer pairs to each junction region defined by nanopore sequencing were used to amplify each specific predicted DVG. These were in turn Sanger-sequenced by primer walking.
  • GI.285 and GI.249 were the two most abundant DVGs at P14, and GI.616 and GI.50 were the most abundant DVGs at P30.
  • GI.285, GI.249, GI.616 and GI.50 all share an identical Nsp1-Nsp10 in-frame junction fragment, which resulted from either a 750/13312 or 751/13313 junction fusion (Fig. 3A).
  • these four DVGs lost substantial ORFIab coding potential as well as all sgRNA coding regions (from S to ORF8) - with the 3’ junction breakpoint occurring within the N gene.
  • GI.249 harbors an additional deletion within the N ORF.
  • Both GI.616 and GI.50 harbor a missense mutation (344CTC346 [Nsp1 - Leu27] to 344TTC346 [Nsp1 - Phe27]) that is not present in the parental genome, nor in GI.285 or GI.249 (Fig. 3A).
  • GI.616 is distinguished from GI.50 in that it harbors a unique 19 amino acid in-frame deletion within Nsp12 (Fig. 3A).
  • RT-qPCR was used to analyze the upstream and downstream junctions (USJ and DSJ, respectively) of the viral RNA isolated from infected cells at different passages.
  • the USJ fragment that was common to GI.285, GI.616 and GI.50, and the DSJ fragment that was characteristic of GI.285 were both highly detected at P14-P16 (Figs. 1 F, and 11).
  • the DSJ fragment that was characteristic of GI.616 and GI.50 (DSJ-3/DSJ-4) emerged later than the USJ (Figs. 3B-3E). Fragments comprising both the USJ and the DSJ of Gl.50/616 became most prominent in the DVG population from P20 to P30 (Figs. 3F-3G).
  • Nanopore sequencing showed a DVG architecture similar to that defined in Exp#1 (Figs. 3K, and 3L).
  • Sanger sequencing confirmed the identical Nsp1-10 breakpoint junction as identified in GI.285, GI.249, GI.616 and GI.50, as well as retention of the frameshift site and Nsp12 coding sequences (Fig. 3A).
  • both the USJ and DSJ fragments characteristic of GI.55 appeared to co-emerge during serial passaging, with both junctions becoming most abundant from P20 - P30 (Figs. 1Q-1S).
  • GI.55 and GI.535 lack the 344 TTC 346 mutation present in GI.616 and GI.50, which further attests to their independent origin (Fig. 3A).
  • DRS revealed a pool of DVG structures at P15 that differed from those seen in the first experiment (compare Fig. 3P, P15 to Fig. 1 D, P14).
  • the two most prominent DVGs at P15 were GI.464 (7.2 kb), and GI.384 (5.7 kb) (Figs. 3Q-3S), and these corresponded in size to the genome species that were detected by Northern blotting in P14 (Fig. 3N, probes B and G, indicated by + and *, respectively).
  • LR-PCR revealed the presence of ⁇ 7 kb genomes that emerged between P11 and P16 and ⁇ 5 kb genomes appearing later between P20 and P30 (Fig. 3U-3V).
  • the abundance of GI.616 and GI.50 was assessed at P20, P25, and P30 and found GI.616 to be the major DVG (25%) present in P25 and P30 (Figs. 3W-3Y).
  • the structure of the DVGs with the highest relative fitness from both these experiments (i) have retained 5' and 3' end sequences, (ii) harbor the identical Nsp1-10 junction breakpoint, and (iii) maintain Nsp11 , the viral frameshift site, and the Nsp12 coding region (Fig. 3Z).
  • a DVG In order to designate a DVG as a DIP, several criteria need to be fulfilled. First, it must be shown that the DVG is capable of interfering with the growth of the parental viral strain. Indeed, for some of the above-mentioned DVGs, this was found to be the case (Figs. 1 B, and 3H). Second, the DVG should be able to undergo packaging in order to be transmitted from cell to cell during virus propagation. This was likely the case for the 5 kb DVGs that emerged at P20 and persisted through serial passaging up to and including P30 (Figs. 1 F-1 G, and 3I-3J).
  • RT-qPCR analysis revealed that only a small amount of cellular GAPDH mRNA was present in the supernatant following infection, which can likely be attributed to cell death (Fig. 4B).
  • SARS-CoV-2 genomic (g)RNA was found to be quite abundant in both supernatant and infected cells.
  • RNA isolated from the cytoplasm of P16- and P30-infected cells as well as in the supernatant of the infected cells (Fig. 4B).
  • Northern blots of RNA from infected cells detected the presence of 5 kb DVGs in cells infected with P30 (Fig. 4G, compare lane 3 to 1 ).
  • the presence of DVGs could also be detected in supernatant obtained from P16- and P30-infected cells (Figs. 4C-4D, compare lanes 5 and 6 to 3).
  • RNA As expected, the control RNA, GAPDH, was detected in cellular RNA preps, but absent from the supernatant samples (Fig. 5C). Also as expected, SARS-CoV-2 gRNA was present in both cytoplasmic and supernatant RNA preps of each of the infected samples, but it was less abundant in the infected P29 samples compared to the P2 samples. While the USJ and DSJ junction fragments common to GI.616 and GI.50 were readily detectable in supernatant and cells that had been infected with at an MOI of 1 the P29 stock, they were absent in the supernatant and cells infected with an MOI of 0.0002 of the same stock (Fig. 5C).
  • DVGs present in P29 could not replicate on their own. Therefore, at this juncture, these DVGs were tentatively classified these as DIP.
  • the prominent DVGs are only propagated in the presence of helper virus, and their detection following serial passaging also indicates that they are packaged. Henceforth, these DVGs are referred to as SARS-CoV-2 DI particles.
  • an EMCV-driven Renilla luciferase (EMCV/Ren) cassette was genetically engineered at the DSJ in GI.55 and GI.616, between Nsp13 and the N ORF (Fig. 6F).
  • EMCV/Ren EMCV-driven Renilla luciferase
  • luciferase activity was also readily detectable, although the synthetic DI RNA/RLuc constructs produced less protein (Fig. 6G). Lysates prepared from P3 and P4 cells showed no activity from serially passaged supernatant from mock- or RLuc mRNA-transfected cells (Fig 6C) In contrast, supernatant from cells having initially received GI.50/RLuc or GI.616/RLuc yielded strong lucifersae activity (Fig. 6G). This was associated with a 13-20 fold reduction in viral titers at P3 and P4 (Fig. 6H). These results indicate that the DI genomes identified herein can be used not only to blunt a SARS-CoV-2 infection but can also be used as a gene delivery vector.
  • EXAMPLE 4 Synthetic, recombinant DI genomes exhibit long-term stability and attenuate SARS-CoV-2 replication.
  • Plaque assays showed that viral titers were reduced by 10- 20-fold in cells that received recombinant DI RNA following infection, whereas no reduction was apparent in cells having received RLuc mRNA (Fig. 7B).
  • Full-length synthetic DI genomes were recovered by LRPCR from RNA of SP4-infected cells (Fig. 70).
  • Probing cell lysates with a-Nsp1 antibodies revealed the presence of Nsp1 in virus infected cells (Fig. 7D, compare lanes 2-4 to lane 1 ).
  • Cells that received the virus from GI.616 transfected cells also expressed an immune-reactive protein whose molecular mass is consistent with it being an Nsp1-10 fusion product (Fig. 70, compare lane 4 to 3).
  • RNA from P0, SP2, and SP4 infections was analyzed by RT-qPCR for the presence of SARS-CoV-2 gRNA and DI genomes (Fig. 7E [raw Ct values] and Fig. 7F [data normalized to GAPDH mRNA levels and expressed relative to CoV-2 gRNA levels]).
  • RLuc mRNA was present in P0 transfected cells, but not in SP2- or SP4-infected cells (Fig. 7E and Fig. 7F).
  • SARS-CoV-2 gRNA was readily detected in all infected cells.
  • the USJ and DSJ, unique characteristics of the DI genomes, were present in transfected (P0) cells, as well as in SP2- and SP4-infected cells (Fig. 7E).
  • EMCV- driven Renilla luciferase (EMCV/RLuc) or transcription regulatory sequence (TRS/RLuc) cassette reporter was inserted into the DSJ of GI.616 (Fig. 7I).
  • Vero E6 cells were infected with SARS-CoV-2, and DI RNA transfections performed 1 hpi. This was then followed by four serial passages. In cells receiving SP4 virus from the RLuc transfections, only background levels of luciferase activity were detected (Fig. 7J). In contrast, cells infected with SP4 virus from GI.616-EMCV/RLuc transfections produced significant luciferase activity (Fig. 7J).
  • GI.616-TRS/RLuc samples which were 320-fold higher than cells containing GI.616-EMCV/RLuc Dis (Fig. 7J).
  • the presence of recombinant GI.616-EMCV/RLuc or GI.616-TRS/RLuc genomes reduced SARS-CoV-2 titers 15- and 30-fold, respectively (Fig. 7K).
  • GI.616-TRS/RLuc produced a subgenomic mRNA containing the viral 5' TRS-L end sequences by RT-PCR using primers targeting TRS-L and the renilla ORF (Fig. 7L, lane 3).
  • synthetic versions of the DI genomes identified herein can be used as conditional gene delivery vectors to inhibit SARS-CoV-2 replication.
  • GI.616 It was next sought to query the mechanism by which GI.616 restricts viral replication. Following infection and transfection of Vero E6 cells, virus was serially passaged three times, and levels of viral RNA in the media and cells were determined at SP2 and SP3 (Fig. 8A). In P2-infected cells, GI.616 reduced SARS-CoV-2 gRNA levels compared to RLuc controls (Figs. 8B-8C). Levels of SARS-CoV-2 gRNA in P2-infected cells relative to virions present in SP3 media were then compared. The presence of GI.616 did not affect the packaging or release of SARS-CoV-2 gRNA from cells (Fig. 8D).
  • GI.616 was packaged and released at the same efficiency as SARS-CoV-2 gRNA (Fig. 8D). The presence of GI.616 did not affect the transmission of SARS-CoV-2 gRNA (Fig. 8E). However, GI.616 genomes transmitted at a rate four-fold higher compared to SARS-CoV-2 gRNA (Fig. 8E). Taken together, these results indicate that robust replication of GI.616 during the early stages of infection (by 4 h) is associated with reduced SARS-CoV-2 gRNA levels over the course of infection.
  • EXAMPLE 6 SARS-CoV-2 Dis encode an Nsp1-10 fusion that inhibits viral replication
  • SARS-CoV-2 Nsp1 is a multifunctional protein that has been implicated in blocking host translation, degradation of cellular mRNAs, and inhibition of nucleo-cytoplasmic mRNA export.
  • Fig. 9A Western blots were performed on extracts from Vero E6 cells infected with P2, P15, or P30 stocks. Results from these experiments showed that Nsp1 (—20 kDa) was present in infected cells (Fig. 9B, bottom arrow), whereas a larger ⁇ 30 kDa protein that cross-reacted with antibodies to Nsp1 was present in P15- and P30-infected cells (top arrow).
  • This protein could also be detected using an antibody targeting the C-terminal domain of Nsp10, which revealed an immunoreactive protein at ⁇ 30 kDa (Fig. 9C, top arrow).
  • the Nsp1-10 fusion protein encoded by the prominent Dis was confined predominantly to the cytoplasm when overexpressed in uninfected cells (Fig. 9D).
  • the Nsp1 C-terminal domain is essential for blocking translation as it interacts with the mRNA entry channel to inhibit cellular protein synthesis. However, this domain is absent from the Nsp1-10 fusion protein (Fig. 9E). Consequently, Nsp1 , but not Nsp1-10, inhibited cellular translation as assessed by polysome profiling (Figs. 9F-9G). Nsp1-10, unlike Nsp1 , did not co-migrate with 40 S ribosomes in polysome gradients (Figs. 9F-9G). These data are consistent with what was observed for a previously described Nsp1(KH/AA) mutant that does not block translation (Figs. 9H-9I).
  • Nsp1 but neither Nsp1-10 nor Nsp1(KH/AA), inhibited 35S-Met/Cys incorporation into the nascent polypeptide chain (Figs. 9J-9K). Nsp1-10 was unable to rescue Nsp1-mediated inhibition of translation in cells (Fig. 9J) or in vitro (Figs. 9L-9M).
  • Nsp1 In vitro, recombinant Nsp1 was shown to be capable of inhibiting cap-dependent translation (Figs. 10A-10F). However, the KH/AA Nsp1 mutant was inactive. Likewise, Nsp1-10 did not display any host translation inhibition activity. The effect of Nsp1-10 was selective in that it did not reduce titers of Dengue type 2 virus (Fig. 10G). The data supports the conclusion that Nsp1-10 is a potent inhibitor of SARS-CoV-2 replication. When Nsp1 and Nsp1-10 were co-transfected into cells, there was no effect on Nsp1 mediated inhibition of translation (Fig. 10H). Lastly, infection of 293/ AC E2 cells stably expressing Nsp1-10 with SARS-CoV2 clearly diminished viral yields 25-fold, relative to control cells (Fig. 101).
  • hACE2 mice were intranasally infected with 5 x 10 4 PFU of stock virus, 5 x 10 4 PFU of P1 virus, 5 x 10 3 PFU of P30-virus, or with a combination of 5 x 10 4 PFU of P1- virus and 5 x 10 3 PFU P30-virus (Fig. 11 A). All animals were monitored daily for clinical symptoms and body weight changes and were sacrificed when body weights dropped ⁇ 20% during the experiment or on day 10. Mice inoculated with stock or P1 virus had to be sacrificed by day 6 post-infection whereas mice receiving only P30 were sacrificed on day 7 (Fig. 11 B).
  • mice inoculated with stock, P1 , or P30 virus attained a grade 4 clinical score (Fig. 11C).
  • Fig. 11C mice receiving the combination of P1 + P30, three were sacrificed on day due to 20% body weight lost but two survived beyond this time point and by day 10 were back to their original weight (Fig. 11 D). None of the five mice receiving P1 + P30 showed a clinical score above 2 (Figs. 11B-11C).
  • EXAMPLE 7 - GI.616 encoded Nsp12 (A19aa) is inactive for polymerase activity.
  • Nsp12 (A19aa) mutation in GI.616 might have on Nsp12 activity was evaluated. Based on the structure of Nsp12, the deletion of 19 amino acids was expected to shorten the distance between the finger region and palm domain of the protein and alter RNA binding (Fig. 12A). Using a polymerase extension assay where activity is assessed using a 4-mer primer, we monitored the appearance of a 14-nts product (Fig. 12B, lane 1 ). The previously described Nsp12 (SNN) active site mutant was inactive in this assay (compare lanes 13-18 to 1 )36.
  • Nsp12(A19aa) mutant was also compromised for polymerase activity (compare lanes 19-24 to 1 ).
  • Nsp1-10 fusion arose in interferon-deficient Vero cells, its function is not restricted to this context since the expression of Nsp1-10 in ACE2-expressing 293 T cells attenuated viral replication (Fig. 9R).
  • the Nsp1-10 protein thus appears to represent a Dl-encoded protein that attenuates helper virus replication.
  • Nsp1-10 did not interfere with cellular translation (Figs. 9F, 9G, and 9J).
  • Nsp1 is a multifunctional protein that has also been implicated in host mRNA cleavage, and blockade of mRNA export.
  • Defective interfering particles can cause cyclical changes in viral titers since they not only compete with but also rely on, parental viruses for propagation.
  • parental virus levels will drop and reach a local minimum, DI levels subsequently do the same as they are dependent on the parental virus for their replication.
  • This leads to parental virus levels peaking as there are minimal DI particles available for competition.
  • this results in cyclic changes in parental virus levels and DI levels where the peak in DI levels is superimposed with a trough in parental virus levels and vice versa.
  • the DI genome replication and encapsidation are two critical parameters that affect wild type virus outcome. Therefore, one successful therapeutic strategy is to deliver the RNA encoding the DI particle intranasally to treat coronavirus infections.

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Abstract

The present invention relates to polypeptides capable of limiting the replication of coronavirus and/ or nucleic acid molecules encoding polypeptides capable of limiting the replication of coronavirus, including those comprising a Nsp1-10 fusion and/or Nsp12. The present invention also relates to methods of using said polypeptides and/or nucleic acid molecules encoding said polypeptides for the treatment coronavirus infections in a subject in need thereof.

Description

POLYPEPTIDES CAPABLE OF LIMITING THE REPLICATION OF A CORONAVIRUS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. provisional application N. 63/290,954 filed December 17, 2021 , incorporated herein by reference in its entirety.
TECHNOLOGICAL FIELD
[0002] The present invention pertains to polypeptides and/or nucleic acids encoding polypeptides that are capable of attenuating coronavirus replication and methods of using same for treating coronavirus infections.
BACKGROUND
[0003] Coronaviruses are a highly diverse family of enveloped positive single-stranded RNA viruses that cause diseases in mammals and birds. They are divided into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus. The seven human coronaviruses have been identified and they all belong to the genera alphacoronavirus or betacoronavirus. These seven coronaviruses primarily cause respiratory symptoms in humans that range from mild to lethal. Gammacoronavirus and deltacoronavirus have no known viruses that infect humans, but contain important agricultural pathogens of livestock.
[0004] Coronaviruses possess remarkably large RNA genomes, which range in size from 27 to 33kb and are flanked by 5’ and 3’ untranslated regions that contain cis-acting secondary RNA structures that are essential for RNA synthesis. In general, the genome of a coronavirus is organized as follows: a 5’-leader-untranslated region (UTR) - Open reading frames (ORF) 1a and 1b - spike (S) - envelope (E) - membrane (M) - nucleocapsid (N) - 3’UTR-poly(A) tail. The ORFs 1a and 1 b, which occupy the first two-thirds of the genome, encode two large overlapping polyproteins, pp1a and pplab. The larger polyprotein, pplab, is a result of a -1 ribosomal frameshift site at the end of open reading frame ORF1a. The ribosomal frameshift allows for the continuous translation of ORF1a followed by ORF1 b. The polyproteins pp1a and pplab have their own proteases that cleave the polyproteins at different sites. The cleavage of polyprotein pplab yields 16 nonstructural proteins (Nsp1 - Nsp16). Nsps 2-16 compose the viral replication and transcription complex that includes, amongst others, RNA-processing and RNA-modifying enzymes and a RNA proofreading function necessary for maintaining the integrity of the coronavirus genome. The ORFs that encode the viral major structural proteins (S, E, M and N) and interspersed ORFs that encode accessory proteins are transcribed from the 3’ one-third of the genome to form a nested set of subgenomic mRNAs. While ORF1a/ORF1ab and the four canonical structural proteins (S, E, M and N) are common to all coronaviruses, the number of accessory proteins and their function is unique depending on the specific coronavirus. The SARS-CoV-2 genome, which is -29.9 kB in size, encodes a total of 27 different proteins.
[0005] Coronavirus infections begin when the spike protein attaches to its complementary host cell receptor. After attachment, a protease of the host cell cleaves and activates that receptor-attached spike protein which allows the virus to fuse with the host cell. The release of the coronavirus genome into the host cell cytoplasm upon entry marks the onset of a complex program of viral gene expression, which is highly regulated in space and time. Translation of ORF1a and ORF1 by the host translational machinery leads to the production of the pp1a and pp1 b polyproteins, which are subsequently cleaved to into the individual Nsps. Many of the Nsps assemble into the replicase-transcriptase complex (RTC) to create environment suitable for RNA synthesis and are ultimately responsible for RNA replication and transcription of subgenomic RNAs. The Nsps also contain other enzyme domains and functions, including those for RNA replication. For example, Nsp1 promotes cellular mRNA degradation and blocks host cell translation and Nsp12 encodes the RNA- dependent RNA polymerase. Following replication and sub-genomic RNA synthesis, the viral structural proteins, S, E and M are translated and moved along to the ER-Golgi immediate compartment (ERGIC). There, viral genomes encapsulated by N protein bud into membranes of the ERGIC containing viral structural proteins, thereby forming mature virions. Following assembly, virions are transported to the cell surface in vesicles and released by exocytosis.
[0006] There is a need for pan-coronavirus therapeutics that target conserved mechanisms utilized by all human coronaviruses and zoonotic coronaviruses. To date, only limited therapeutic options are available to prevent and treat specific coronavirus infections. With the exception of the nucleotide analogue prodrug remdesivir, and despite several efforts, there is no known specific, proven, antiviral treatment capable of efficiently and rapidly inducing viral containment and clearance of SARS-CoV-2, as well as no broadspectrum drug for other human pathogenic coronaviruses. Given that most adverse outcomes of coronavirus disease are associated with severe inflammation (ie. a cytokine storm), corticosteroid drugs (e.g. dexamethasone) have been administered systemically to severely ill Covid-19 patients in a hyper-inflammatory state. However, the use of systemic corticosteroids in treating severe coronavirus infections is controversial due to the side effects associated with their use. Further, if they are administered at the wrong time during the infection (ie. during the early phase of infection) corticosteroids might actually allow increased viral replication and aggravate the disease. Vaccines have long been considered the gold standard for infectious disease prevention and eradication. However, zoonotic pathogens like coronaviruses emerge from animal reservoir species, thus vaccination strategies are unlikely to lead to eradication while the virus continues to circulate in reservoir hosts. Monoclonal antibodies (mAbs) have potential utility in combating viral diseases caused by coronavirus, by prophy lactically and therapeutically neutralizing structural proteins on the outside of the virion. Indeed, mAbs have been shown to improve survival in patients hospitalized with COVID-19 who were unable to mount effective immune responses to the virus on their own. Further, mAbs may also have a role in preventing infections, particularly in unvaccinated close contacts of individuals who are known to be infected with a coronavirus. However, one drawback of mAb therapy is that, due to differences in the structural proteins between different coronavirus species, separate formulations of mAbs are likely to be required for different coronavirus species and/or strains. Additionally, it may be possible for coronaviruses to evolve mutations that allow them to escape neutralization by the mAb.
[0007] It would be highly desirable to be provided with polypeptides that can limit coronavirus replication. It would also be highly desirable to be provided with methods for using said molecules for treating coronavirus infections in a subject in need thereof.
BRIEF SUMMARY
[0008] The present disclosure concerns isolated polypeptides, isolated nucleic acid molecules and/or therapeutic interfering particles derived from coronaviruses that are capable to limiting coronavirus replication.
[0009] According to a first aspect, the present disclosure provides an isolated polypeptide capable of limiting the replication of a coronavirus, whereby the isolated polypeptide comprises a fusion of at least two polypeptides encoded by viral open reading frames of a ORF1a of a coronavirus. In some embodiments, the two polypeptides are Nsp1 and Nsp10 and the fusion polypeptide comprises a Nsp1 moiety and a Nsp10 moiety. In some embodiments, the Nsp1 moiety of the fusion protein comprises the amino acid sequence of SEQ ID NO:3, a variant of the amino acid sequence of SEQ ID NO: 1 or 3 and/or a fragment of the amino acid sequence of SEQ ID NO: 1 or 3. In some embodiments, the Nsp10 moiety of the fusions protein comprises the amino acid sequence of SEQ ID NO:4, a variant of the amino acid sequence of SEQ ID NO: 2 or 4 and/or a fragment of the amino acid sequence of SEQ ID NO: 2 or 4. In other embodiments, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 5, a variant of the amino acid sequence of SEQ ID NO: 5 and/or a fragment of SEQ ID NO:5. [0010] According to a second aspect, the present disclosure provides an isolated nucleic acid molecule encoding the fusion polypeptides described herein. In some embodiments, the isolated nucleic acid molecule comprises one or more ribonucleic acid residues. In other embodiments, the isolated nucleic acid molecule is a messenger RNA (mRNA) molecule. In still other embodiments, the isolated nucleic acid molecule comprises one or more deoxyribonucleic acid (DNA) residues. In yet other embodiments, the isolated nucleic acid molecule is a DNA molecule. In some embodiments, the isolated nucleic acid molecule further comprises a heterologous gene.
[0011] According to a third aspect, the present disclosure provides an isolated nucleic acid molecule comprising a defective viral genome (DVG) encoding a therapeutic interfering particle (TIP), wherein the TIP: (i) is capable of limiting the replication of a human coronavirus, (ii) is replication defective and can be replicated in the presence of a helper virus; (iii) is defective for packaging and can be packaged in the presence of a helper virus,
(iv) is capable of being enriched upon a plurality of passage in a cell infected with the coronavirus and/or the helper virus at a multiplicity of infection equal to or greater than 1 ; and
(v) encodes the isolated polypeptide of the present disclosure and/or the nucleic acid molecule encoding the isolated peptide which optionally comprises one or more ribonucleic acid residues, (e.g. is a mRNA), or optionally comprises one or more deoxyribonucleic acid residues (e.g. is a DNA). In some embodiments, the TIP encoded in the DVG is a defective interfering particle (DIP). In some embodiments, the coronavirus is the helper virus. In some embodiments, the human coronavirus and/or the helper virus is from the alpha genus. In other embodiments, the human coronavirus and/or the helper virus is 229E or NL63. In yet other embodiments, the human coronavirus and/or the helper virus is from the beta genus. In further embodiments, the human coronavirus and/or the helper virus is 0043, HKU1 , SARS- CoV, MERS-CoV, or SARS-CoV2. In yet other embodiments, the human coronavirus and/or helper virus is from the gamma genus. In other embodiments, the human coronavirus and/ or helper virus is from the delta genus. In some embodiments, the isolated nucleic acid molecule comprising a defective viral genome (DVG) may comprise, when compared to the nucleic acid sequence of the coronavirus or of the helper virus, a first deletion in an open reading frame 1a (ORF1a). In other embodiments, the isolated nucleic acid molecule contains a first deletion at a first start position corresponding to position 749 of GenBank accession number NC_045512. In some embodiments, the first deletion ends at the first position corresponding to position 13311 of GenBank accession number NC_045512. In some embodiments, the first deletion in an ORF1a encompasses the nucleic acid sequence encoding at least one of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, or Nsp9. In some embodiments, the TIP encoded in the nucleic acid molecule comprising a DVG is capable of limiting the titer of the coronavirus and/or helper virus by at least one log or more. In other embodiments, the isolated nucleic acid molecule has a nucleic acid sequence of GI.285 (SEQ ID NO:6), GI.249 (SEQ ID NO:7), GI.616 (SEQ ID NO:8), GI.50 (SEQ ID NO:9), GI.55 (SEQ ID NO:10) or GI. 535 (SEQ ID NO:11 ).
[0012] In a fourth aspect, the present disclosure provides a TIP comprising the polypeptide defined in any one of the first aspect and/or the nucleic acid molecule of the second aspect.
[0013] In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising (i) the polypeptide of the first aspect and/ or the isolated nucleic acid molecule of the second aspect and (ii) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for nasal administration.
[0014] In a sixth aspect, the present disclosure provides a method for treating a coronavirus infection in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the polypeptide of the first aspect, the isolated nucleic acid of the second aspect and/or the pharmaceutical composition of the fifth aspect, to the subject so as to reduce the replication of the coronavirus. In one embodiment, the subject is a human subject.
[0015] In a seventh aspect, the present disclosure provides the use of a therapeutically effective amount of the polypeptide of the first aspect, the isolated nucleic acid of the second aspect and/or the pharmaceutical composition of the fifth aspect, for treating a coronavirus infection in a subject in need thereof. In one embodiment, the subject is a human subject.
[0016] In an eighth aspect, the present disclosure provides the polypeptide of the first aspect, the isolated nucleic acid of the second aspect, and/or the pharmaceutical composition of the fifth aspect, for treating a coronavirus infection in a subject in need thereof. In one embodiment, the subject is a human subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:
[0018] Fig. 1A is schematic diagram illustrating a strategy to generate SARS-CoV-2 DIPs in Vero E6 cells. Northern blotting and nanopore direct (or native) RNA sequencing (sequencing of RNA molecules without conversion to DNA, currently only achieved by Nanopore sequencing) were used to assess the emergence of prominent DVGs during serial passaging. [0019] Fig. 1B is a graph showing the Quantitation of virus titers obtained from the indicated passages (P), n=3 ± standard deviation (SD).
[0020] Fig. 1C is a photograph of a representative set of plaque assays performed on Vero E6 cells with supernatant taken at the indicated passages.
[0021] Fig. 1 D shows a genome coverage of nanopore RNA sequencing data from P1 , P14, and P30. The “step” changes (indicated by upward arrows in P1) occur at the 5' borders of the S, 3a, E, 6, and N sgRNAs. The reference genome position (nt) is shown at the bottom.
[0022] Fig. 1 E is a schematic diagram of the SARS-CoV-2 genome illustrating ORF organization. The boxes with nucleotide coordinates and upper-case letters denote location of 32P-labelled probes (A to G) used for Northern blotting.
[0023] Fig. 1 F is a Northern blot analysis performed on RNA isolated from Vero E6 cells infected with SARS-CoV-2 from the indicated passages after a short exposure (S.E) (2 h/RT). Plus sign, asterisks, and arrow highlight prominent 5 kb DVGs emerging from P20- P30.
[0024] Fig. 1G is a Northern blot analysis performed on RNA isolated from Vero E6 cells infected with SARS-CoV-2 from the indicated passages after to long exposure (L.E.) (3.5 h/- 70°C with intensifying screen). Plus sign, asterisks, and unlabelled arrow highlight prominent 5 kb DVGs emerging from P20-P30.
[0025] Fig. 1 H shows genome coverage of nanopore RNA sequencing data from P1 , P15, and P29. The “step” changes (indicated by upward arrows in P1 ) occur at the boundary of the S, 3a, E, 6, and N ORFs and are reflective of the 5’ borders of the major sgRNAs. The reference genome position (nt) is shown at the bottom.
[0026] Fig. 11 shows the architecture of the top 50 most abundant DVGs from P15 infected cells, including the percentage of read counts corresponding to the transcript model. The “% of Transcript Model Reads” corresponds to the number of viral reads mapped to a collapsed transcript model (as shown for individual DVGs plotted to the right) divided by the total number of all viral RNA reads x 100. Open colored boxes are retained sequences and thin lines correspond to deletions. The CoV-2 reference genome is shown above the DVGs, along with the encoded polypeptides. Nucleotide position is shown below.
[0027] Fig. 1J shows the architecture of the top 50 most abundant DVGs from P29 infected cells, including the percentage of read counts corresponding to the transcript model. The “% of Transcript Model Reads” corresponds to the number of viral reads mapped to a collapsed transcript model (as shown for individual DVGs plotted to the right) divided by the total number of all viral RNA reads x 100. Open colored boxes are retained sequences and thin lines correspond to deletions. The CoV-2 reference genome is shown above the DVGs, along with the encoded polypeptides. Nucleotide position is shown below.
[0028] Fig. 1 K is a Northern blot analysis performed on RNA isolated from the indicated passages of SARS-CoV-2-infected cells with Nsp12.
[0029] Fig. 1L is a Northern blot analysis performed on RNA isolated from the indicated passages of SARS-CoV-2-infected cells with ORF10+3’UTR.
[0030] Fig. 1 M shows the architecture of the top seven most abundant DVGs obtained from R1 -infected cells from Exp #1 having retained 5' and 3' end sequences. The SARS- CoV-2 reference genome is shown at the top, along with the encoded polypeptides.
[0031] Fig. 1 N shows the architecture of the top seven most abundant DVGs obtained from P14 -infected cells from Exp #1 having retained 5' and 3' end sequences. The SARS- CoV-2 reference genome is shown at the top, along with the encoded polypeptides.
[0032] Fig. 10 shows the architecture of the top seven most abundant DVGs obtained from P30 -infected cells from Exp #1 having retained 5' and 3' end sequences. The SARS- CoV-2 reference genome is shown at the top, along with the encoded polypeptides.
[0033] Fig. 1 P is a schematic showing the location of primers used for amplification (A1/A2) and for assessing presence of USJ (US-J/1/USJ-2) and DSJ (DSJ-3/DSJ-4) fragments in GI.535. Shown are the nucleotide sequenc es flanking the breakpoints in GI.535.
[0034] Fig. 1Q is an image of an end-point polymerase chain reaction (PGR) gel showing emergence of most prominent DVGs at P20 with stable maintenance to P30. Amplifications were performed using primers MIK2. Products were obtained following 30 amplification cycles and analyzed on a 0.8% agarose/TAE gel.
[0035] Fig. 1 R is a graph showing amplification analysis of DVG breakpoints using USJ primer pairs. RNA from infected cells of the indicated passages was reverse transcribed and cDNA used as template for qPCRs. The relative abundance of each PGR product was calculated using the ACt method and set relative to GAPDH. n=2.
[0036] Fig. 1S is a graph showing amplification analysis of DVG breakpoints using DSJ primer pairs. RNA from infected cells of the indicated passages was reverse transcribed and cDNA used as template for qPCRs. The relative abundance of each PGR product was calculated using the ACt method and set relative to GAPDH. n=2. [0037] Fig. 1T is a pie chart illustrating relative abundance of DVGs in P20 from Experiment 1.
[0038] Fig. 1U is a pie chart illustrating relative abundance of DVGs in P25 from Experiment 1.
[0039] Fig. 1V is a pie chart illustrating relative abundance of DVGs in P30 from Experiment 1.
[0040] Fig. 2A is a schematic showing the location of primers used for long range PGR amplification are indicated. Complementary DNA was prepared using oligo d(T) and primers A1 and A2 were used in the long-range PCRs. Junction primers were designed based on direct RNA nanopore sequencing and used to amplify the corresponding genomes from the long-range PCR products.
[0041] Fig. 2B is an image of a gel showing PCR products obtained with primers A1 and A2 using LA-Taq hot start DNA Polymerase (Takara) following long-range amplification from RNA isolated from uninfected Vero E6 cells (Mock) or Vero E6 cells infected with the indicated viral passages. H2O sample contained no input RNA in RT reaction.
[0042] Fig. 2C is an image of a gel showing PCR products obtained using the indicated junction primer pair B1/B2 and the PCR product generated in Fig. 2B as template. Arrows indicate PCR products obtained of the expected molecular mass.
[0043] Fig. 2D is an image of a gel showing PCR products obtained using the indicated junction primer pair B1/C1 and the PCR product generated in Fig. 2B as template. Arrows indicate PCR products obtained of the expected molecular mass.
[0044] Fig. 2E is shematic showing the location of primers used for long range PCR amplification. The difference between Gl. 616 and GI.50 is a 19 amino acid in-frame deletion in Nsp12.
[0045] Fig. 2F is an image of a gel of PCR products obtained following long-range amplification from RNA isolated from Vero E6 cells infected with the indicated viral passages. H2O samples contained no input RNA in RT reaction, M (NEB 1 kb DNA ladder). Arrow indicates PCR product that was gel purified and Sanger sequenced by primer walking. Primers spanning or unique to the Nsp12 internal deletion were used to distinguish GI.616 from GI.50, respectively.
[0046] Fig. 2G is a sequencing chromatogram showing the 5’ Nsp1/10 (750/13312 or 751/13313) junction fragment obtained by Sanger sequencing. [0047] Fig. 2H is a sequencing chromatogram showing the 3’ Nsp13/N (16829/29446) junction fragment obtained by Sanger sequencing.
[0048] Fig. 3A shows a genome architecture of the two most prevalent DVGs present in infected Vero E6 at P15 and P29 (Exp#1 ) and at P30 (Exp #2). Nucleotide position is based on the SARS-CoV-2 Wuhan-Hu-1 isolate (NC_045512.2). Asterisks denotes a missense mutation converting 344CTC346 to 344TTC346 in Nsp1.
[0049] Fig. 3B shows a schematic representation of the structure of the indicated DVGs. Right angled arrows indicate location of primers used for qPCR analysis.
[0050] Fig. 3C is a graph showing the RT-qPCR analysis of DVG breakpoints using USJ-1/USJ-2 primer pair. RNA from infected cells of the indicated passages was reverse transcribed with random hexamers and cDNA used as template for qPCRs. The relative abundance of each PCR product was calculated using the AACt method and set relative to GAPDH, n=2.
[0051] Fig. 3D is a graph showing the RT-qPCR analysis of DVG breakpoints using DSJ-1/DSJ-2 primer pair. RNA from infected cells of the indicated passages was reverse transcribed with random hexamers and cDNA used as template for qPCRs. The relative abundance of each PCR product was calculated using the AACt method and set relative to GAPDH, n=2.
[0052] Fig. 3E is a graph showing the RT-qPCR analysis of DVG breakpoints using DSJ-3/DSJ-4 primer pair. RNA from infected cells of the indicated passages was reverse transcribed with random hexamers and cDNA used as template for qPCRs. The relative abundance of each PCR product was calculated using the AACt method and set relative to GAPDH, n=2.
[0053] Fig. 3F is an image of a gel obtain for end-point PCR products (P2 to P8). Amplifications were performed using primers USJ-1 and DSJ-4. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
[0054] Fig. 3G is an image of a gel obtain for end-point PCR products (P9 to P30). Amplifications were performed using primers USJ-1 and DSJ-4. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
[0055] Fig. 3H is a graph showing the quantitation of virus titers obtained from the indicated passages. n=3 ± SD.
[0056] Fig. 3I is a Northern blot analysis performed on RNA isolated from the indicated SARS-CoV-2 infected cells with probe B. Arrow highlights prominent DVGs emerging at late passages. [0057] Fig. 3J is a Northern blot analysis performed on RNA isolated from the indicated SARS-CoV-2 infected cells with probe G. Arrow highlights prominent DVGs emerging at late passages.
[0058] Fig. 3K shows the characterization of DVGs obtained in Exp#2 by nanopore direct RNA sequencing. Genome coverage of the nanopore direct RNA sequencing data from P1 , P14, and P30. The “step” changes (indicated by upward arrows in P1 ) occur at the boundary of the S, 3a, E, 6, and N ORFs and are reflective of the 5’ borders of the major sgRNAs.
[0059] Fig. 3L shows the architecture of the top 50 most abundant DVGs from P30 infected Vero cells. DVGs from P30 in Vero cells (Expt#2) were used. The I percentage of read counts corresponding to each transcript model is shown.
[0060] Fig. 3M is a schematic diagram of the SARS-CoV-2 genome illustrating ORF organization. The boxes with nucleotide coordinates and upper-case letters denote location of 32P-labelled probes used for Northern blotting.
[0061] Fig. 3N is a Northern blot analysis performed on intracellular RNA isolated from the indicated passages. RNA markers (NEB) are indicated to the left and size distribution is presented in kilobases (kb). The assignment of sgRNAs is based on predicted size. The plus sign and asterisks highlight DVGs present in P14. The same Northern blot was used in all probings shown in this panel. GAPDH was used to assess mRNA quality. Mock, uninfected cells; gRNA, genomic RNA.
[0062] Fig. 30 is a Northern blot analysis performed on RNA isolated from SARS-CoV-2 infected cells at the indicated passages.
[0063] Fig. 3P shows the genome coverage of nanopore DRS data from P1 , P15 and P29. The “step” changes (indicated by upward arrows in P1 ) occur at the 5’ border of the S, 3a, E, 6, and N sgRNAs.
[0064] Fig. 3Q shows the architecture of the top 7 most abundant DVGs from P1 infected cells obtained in Exp#2 and that had retained 5’ and 3’ end sequences, the read counts corresponding to the transcript model are also shown.
[0065] Fig. 3R shows the architecture of the top 7 most abundant DVGs from P15 infected cells obtained in Exp#2 and that had retained 5’ and 3’ end sequences, the read counts corresponding to the transcript model are also shown.
[0066] Fig. 3S shows the architecture of the top 7 most abundant DVGs from P29 infected cells obtained in Exp#2 and that had retained 5’ and 3’ end sequences, the read counts corresponding to the transcript model are also shown. [0067] Fig. 3T shows the architecture of GI.616 and GI.50 in an inverted black triangle denoting a 19 amino acid deletion in GI.616. GI.50 and GI.616 which harbor identical US and DS junctions.
[0068] Fig. 3U is an image of end-point PGR showing emergence of most prominent DVG (P2-P8). Amplifications were performed using primers A1 and A2. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
[0069] Fig. 3V is an image of end-point PGR showing emergence of most prominent DVG (P9-P30). Amplifications were performed using primers A1 and A2. Products were obtained following 30 amplification cycles and analyzed on a 0.7% agarose/TAE gel.
[0070] Fig. 3W is a pie chart illustrating the relative abundance of DVGs in P20 from Exp#2.
[0071] Fig. 3X is a pie chart illustrating the relative abundance of DVGs in P25 from Exp#2.
[0072] Fig. 3Y is a pie chart illustrating the relative abundance of DVGs in P30 from Exp#2.
[0073] Fig. 3Z shows the genome architecture of the most prevalent DVGs isolated from infected cells at P30. Nucleotide position is based on the SARS-CoV-2 Wuhan-Hu-1 isolate (NC_045512.2).
[0074] Fig. 4A is a schematic showing that DVGs are packaged and present in extracellular supernatant. Vero E6 cells were infected with virus from P1 , P16, or P30 and after 24 h, RNA was isolated from the supernatant or infected cells for analysis.
[0075] Fig. 4B shows RT-qPCR analysis of RNA isolated from supernatant (S/N) or the cytoplasm (Cyto) of infected cells at the indicated passages. Raw Ct values are presented, n=3 ± SD
[0076] Fig. 4G is a Northern blot analysis of cellular RNA from the indicated sources and viral passages. Exposure time for the blot probing cellular RNA (lanes 1-3) was 3 days (- 70°C/intensifying screen).
[0077] Fig. 4D is a Northern blot analysis of S/N RNA from the indicated sources and viral passages. Exposure time for the blot probing the supernatant (S/N) RNA (lanes 4-6) was 14 days (-70°C/intensifying screen).
[0078] Fig. 5A is a schematic diagram showing DVG-dependency on parental virus for replication and propagation. At an MOI = 1 , both parental and DI genomes are expected to be maintained upon serial passage. [0079] Fig. 5B is a schematic diagram showing DVG-dependency on parental virus for replication and propagation. At low MOI (0.0002) in which parental genomes and Dis enter different cells, the DI will be lost upon sequential serial passaging.
[0080] Fig. 5C is a graph showing a RT-qPCR analysis of RNA isolated from cells or supernatant infected with the indicated viral passages and MOI. n =3 biologically independent experiments ± SD.
[0081] Fig. 5D is an image of gel from amplification products of Dis from Vero cell lysates and media that had been infected with the indicated viral stocks at an MOI of 1 or 0.0002. Amplifications were performed using A1 and A2 primers for 30 cycles. Products were analyzed on a 0.8% agarose/TAE gel. White arrows indicate recovery of 5 kb DVGs. M; 1 kb DNA ladder.
[0082] Fig. 6A shows an experimental flow used to generate synthetic DI particles. Following infection of Vero E6 cells with SARS-CoV-2 at an MOI = 1 , cells were transfected with in vitro synthesized Renilla luciferase (RLuc) mRNA, GI.50, GI.55, or GI.616 RNA eight hours post-infection (hpi). Supernatant was collected 22 h later, clarified, and used to infect a new set of Vero E6 cells four subsequent times. The location of the upstream junction (USJ) and the downstream junction (DSJ) primers is indicated on the map of the synthetic template.
[0083] Fig. 6B is a graph showing the RT-qPCR analysis of RNA from P0, P2, and P4 infected cells. RNAs targeted by each oligo pair is shown on the bottom. Obtained Ct values are displayed, n=2 ± SD.
[0084] Fig. 6C shows results after Vero E6 cells were transfected with H2O (control), GI.50, GI.55, or GI.616 and cell media was collected 22 h later, clarified and used to “infect” new cells. This was repeated one more time (P2 cells). RT-qPCR analysis of RNA from P0 (transfected) and P2 cells. RNAs targeted by each oligo pair is shown on the bottom. Obtained Ct values are displayed, n=2 ± SD.
[0085] Fig. 6D is a an image of a gel showing RT-Long range PCR showing recovery of ID genomes from P4 infected cells. RNA from the indicated samples were used to generate cDNA using random primers, followed by long range PCR amplification.
[0086] Fig. 6E is a graph showing the quantitation of virus titers obtained from the indicated DIPs at P4. n=3 ± SD.
[0087] Fig. 6F is schematic diagram of DI genomes harboring EMCV/RLuc expression cassette.
[0088] Fig. 6G is a graph showing the luciferase activity obtained from cells mock transfected or transfected with Ren, GI.55-EMC/RLuc and GI.616-EMC/RLuc mRNA. Extracts were prepared from cells that had been mock infected (mock) or received SARS- CoV-2 virus. The supernatant was harvested 24 hpi (PO) or after 1 to 4 serial passages of the viral supernatant obtained from PO cells (labelled P1 - P4).
[0089] Fig. 6H is a graph showing SARS-CoV-2 virus titers obtained from P3 or P4, n=3 ± SD.
[0090] Fig. 7A is a schematic of the experimental flow used to generate synthetic DI particles. Following infection of Vero E6 cells with SARS-CoV-2 at an MOI =1 , cells were transfected with in vitro synthesized RLuc, GI.50, or GI.616 RNA. Media was collected 22 h later, clarified, and used in serial infections (four passages) of Vero E6 cells.
[0091] Fig. 7B is a graph showing the quantification of virus titers obtained from the indicated Dis at SP4. n= 3 biologically independent experiments ± SD. ns nonsignificant — p > 0.9 (two-way ANOVA).
[0092] Fig.7C is an image of a gel showing recovery of DI genomes from SP4-infected cells.
[0093] Fig. 7D is a Western blot of extracts probed with a-Nsp1 or a-actin antibodies. Lysates analyzed were prepared from uninfected (mock) Vero E6 cells (lane 1 ) or Vero cells receiving SP2 from untransfected cells (-) (lane 2), RLuc mRNA-transfected cells (lane 3), or GI.616 RNA-transfected cells (lane 4). Dotted arrow denotes Nsp1 and red arrow denotes Nsp1-10 fusion.
[0094] Fig. 7E is a graph showing RT-qPCR analysis of RNA from P0, SP2-, and SP4- infected cells. RNAs targeted by each oligo pair is shown on the bottom. Obtained Ct values are displayed, n = 2 biologically independent experiments, black bar represents the mean.
[0095] Fig. 7F is a graph showing a RT-qPCR analysis of RNA from P0, SP2, and SP4 infected cells. RLuc, DI USJ and DSJ, and CoV-2 gRNA RNA levels were calculated as a fold change relative to GAPDH using the 2-ACT method. Values corresponding to the USJ and DSJ junction were averaged to yield a final DI level. Lastly, RLuc and DI levels were expressed relative to CoV-2 gRNA levels.
[0096] Fig. 7G is a graph showing RT-qPCR analysis of RNA from P0, SP2, and SP4 infected cells. The identity of RNAs targeted by each oligo pair is shown at the bottom. Obtained Ct values are displayed. n=2.
[0097] Fig. 7H is a graph showing the results after Vero E6 cells were transfected with GI.50 or GI.616 RNA, media collected 22 h later, clarified and applied to fresh cells for two serial passages. RT-qPCR analysis of 18S rRNA, SARS-CoV-2 gRNA, and the Dl-specific USJ and DSJ sites from uninfected PO and SP2 cells. RNA or regions targeted by each oligo pair is shown at the bottom. Obtained Ct values are displayed. n=2.
[0098] Fig. 7I is a schematic diagram of DI genomes harboring EMCV/RLuc or TRS/RLuc expression cassettes.
[0099] Fig. 7J is a graph showing RLuc activity obtained from the indicated constructs at SP4. n = 4 biologically independent experiments ± SD.
[00100] Fig. 7K h is a graph showing the quantification of virus titers obtained from the indicated Dis at SP4. n = 4 biologically independent experiments ± SD.
[00101] Fig. 7L is an image of a gel of a RT-PCR showing the presence of an RLuc sgRNA containing sequences upstream of the 5' TRS-L site in SARS-CoV-2-infected cells transfected with GI.616-TRS/RLuc.
[00102] Fig. 8A is a schematic diagram showing the experimental design for assessing the effect of GI.616 on SARSCoV- 2 replication. SARS-CoV-2 and GI.616 genomes were isolated from P2 and P3 cells, as well as SP3 supernatant.
[00103] Fig. 8B is a graph showing the growth rates (absolute gRNA levels relative to the amount at 4 h) of parental virus propagated in the presence of GI.616 (+) or RLuc (-). The data for two independent experiments is shown (Exp1 and Exp4).
[00104] Fig. 8C is a graph showing the growth rates (absolute gRNA levels relative to the amount at 4 h) of parental virus propagated in the presence of GI.616 (+) or RLuc (-). The data for two independent experiments is shown (Exp2 and Exp3).
[00105] Fig. 8D is a graph showing the percent packaged genomes upon propagation of SARS-CoV-2 in the presence or absence of GI.616. n = 4 independent biologically experiments ± SD. "ns” means not significant (p= 0.25).
[00106] Fig. 8E is a graph showing the transmission efficiency of SARS-CoV-2 and GI.616. n = 4 independent biological experiments ± SD.
[00107] Fig. 9A is schematic showing the coding potential of Dis. Black triangle indicates the 19 amino acid deletion in GI.616.
[00108] Fig. 9B is a Western blot of extracts probed with a-Nsp1 antibodies. Lysates analyzed were prepared from uninfected (mock) Vero E6 cells or Vero E6 cells infected with P2, P15, and P30 (Exp #2) viral stocks. Dotted arrow denotes Nsp1, filled arrow denotes arrow denotes Nsp1-10 fusion.
[00109] Fig. 9C is a Western blot of extracts probed with a-Nsp10 C-terminal domain antibodies. Lysates analyzed were prepared from uninfected (mock) Vero E6 cells or Vero E6 cells infected with P2, P15, and P30 (Exp #2) viral stocks. Unnbelled arrow denotes Nsp1-10 fusion.
[00110] Fig. 9D is an image of a gel demonstrating that Nsp1-10 is predominantly a cytoplasmic protein. pcDNA-based expression vectors were transfected into 293T cells, and 48 h later cells were harvested in PBS. Following subcellular fractionation (C, cytoplasm; N, nuclear), proteins were resolved on a 10% SDS-polyacrylamide gel, transferred to immobilon PVDF membrane, and probed with antibodies indicated to the right. eEF2 and hnRNPAI were used as loading controls for cytoplasmic and nuclear fractions, respectively.
[00111] Fig. 9E is a Clustal Omega alignment of Nsp1 and Nsp1-10 fusion. The extent of two Nsp1 deletion mutants are indicated by dotted lines. The location of the Nsp1 KH amino acids that were mutated to AA are indicated.
[00112] Fig. 9F is a graph showing a polysome analysis of 293 T cells transfected with the indicated expression vectors (20 pg). Cytoplasmic extracts were prepared 24 h posttransfections and polysomes analyzed by sucrose gradient sedimentation.
[00113] Fig. 9G is a Western blot analysis undertaken on the protein samples obtained from individual polysome fractions as per Fig. 9F. Western blots were probed with antibodies shown to the right.
[00114] Fig. 9H is a graph showing a polysome analysis of 293T cells transfected with FLAG-Nsp1 (KH/AA) expression vector (10 pg). Cytoplasmic extracts were prepared 24h post-transfections and polysomes analyzed by sucrose gradient sedimentation.
[00115] Fig. 9I is a Western blot analysis of protein samples obtained from individual fractions as per Fig. 9H. Fraction numbers are indicated. Western blots were probed with antibodies shown to the right.
[00116] Fig. 9J is a graph showing that the ectopic expression of Nsp1-10 does not inhibit translation in 293 T cells. 293 T cells were transfected with the indicated amounts of expression vector. Twenty-four hours later, cells were metabolically labeled with 35S- Met/Cys for 15 min. TCA precipitation was used to determine the amount of radiolabel incorporated into proteins and counts were normalized to total protein content in the extract and expressed relative to cells having received empty vector (pcDNA3; which was set to 1). n = 4 biologically independent experiments ± SD. ns, p > 0.05; *, 0.01 > p > 0.05 (Dunnett’s multiple comparisons test).
[00117] Fig. 9K is a Western blot analysis of protein samples obtained in Fig. 9J. Protein extracts were prepared from cells transfected with the indicated expression vectors, resolved on a 10% SDS-polyacrylamide gel, and transferred to immobilon PVDF mem branes. Western blots were probed with antibodies shown to the right.
[00118] Fig. 9L is a coomassie stain of 10% SDS-PAGE of purified recombinant protein used in in vitro translation assays.
[00119] Fig. 9M is a graph showing in vitro inhibition of translation by recombinant Nsp1 , but not Nsp1-10. Relative luciferase values obtained following in vitro translations of RRL programmed with 20 pg/mL FF/HCV/Ren mRNA and supplemented with the indicated amounts of recombinant protein. RRL was pre-incubated with recombinant protein for 5 mins at 30° C before the addition of FF/HCV/Ren mRNA. Luciferase values are set relative to those obtained from extracts that received only protein storage buffer. n= 3 ± SD.
[00120] Fig. 9N is a graph showing RT-qPCR analysis of RNA from P2 cells infected with the indicated Dis. RNA preps targeted by each oligo pair is shown at the bottom. Obtained Ct values are displayed, n = 3 biologically independent experiments ± SD.
[00121] Fig. 90 is a graph showing a RT-qPCR analysis of RNA from SP4 infected cells. For each of the transfections, the DI USJ and DSJ, and CoV-2 gRNA RNA levels were calculated as a fold change relative to GAPDH using the 2-ACT method. For Nsp1-10 A 2NTD, only DSJ levels were assessed due to absence of an USJ. The values corresponding to the USJ and DSJ were averaged to calculate DI levels. Finally, DI levels were expressed relative to CoV-2 gRNA levels.
[00122] Fig. 9P is graph showing the quantification of virus titers obtained with the indicated Dis at P4. n =3 biologically independent experiments ± SD.
[00123] Fig. 9Q is a Western blot of 293 T/ACE2 cells stably expressing BirA (Ctrl, control) or Nsp1-10.
[00124] Fig. 9R is graph showing SARS-CoV-2 virus titers obtained in 293 T/ACE2/BirA or 293 T/ACE2/Nsp1-10 cells, n = 3 biologically independent experiments ± SD.
[00125] Fig. 10A is a graph showing a polysome analysis of 239T cells transfected with FLAG-NSp1 vector (30 pg). Cytoplasmic extracts were prepared 24 h post-transfections and polysomes analyzed by sucrose gradient sedimentation.
[00126] Fig. 10B is a graph showing a polysome analysis of 239T cells transfected with FLAG-NSp1 vector (KH/AA) (30 pg). Cytoplasmic extracts were prepared 24 h posttransfections and polysomes analyzed by sucrose gradient sedimentation.
[00127] Fig. 10C is a Western blot analysis of protein samples obtained from individual fractions in Fig. 10A. Fraction numbers are indicated to the top. [00128] Fig. 10D is a Western blot analysis of protein samples obtained from individual fractions in Fig. 10B. Fraction numbers are indicated to the top.
[00129] Fig. 10E is a Coomassie stain of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of purified recombinant protein used in in vitro translation assays.
[00130] Fig. 10F is a graph showing in vitro inhibition of translation by recombinant Nsp1 , but not Nsp1-10. The top panel shows a schematic diagram of FF/HCV/Ren mRNA reporter. The figure shows the relative luciferase values obtained following in vitro translations of RRL programmed with 10 g/ml FF/HCV/Ren mRNA and supplemented with the indicated amounts of recombinant protein. RRL was pre-incubated with recombinant protein for 5 mins at 30°C before the addition of FF/HCV/Ren. Luciferase values are set relative to those obtained from extracts that received only protein storage buffer, n= 3 ± SD.
[00131] Fig. 10G is a graph showing Dengue type 2 virus titers obtained in 293T/ACE2/BirA (Ctrl) or 293T/ACE2/Nsp1-10 (Nsp1-10) cells. n=3 ± SD, ns - not significant.
[00132] Fig. 10H is a Western blot of extracts prepared from the indicated cells demonstrating the expression of recombinant Nsp1-10 in 293T/ACE2 cells.
[00133] Fig. 101 is a graph demonstrating the reduction in virus titers obtained following infection of 293T/ACE2(Nsp1-10) cells, relative to titers obtained in 293T/ACE2 cells.
[00134] Fig. 11A is a shematic showing cohorts of C57BL/6 hACE2 mice intranasally infected with the indicated doses of stock, P1 , or P30 virus.
[00135] Fig. 11 B is a graph showing the change in body weight for the mice of Fig. 10A. The dotted line represents the cutoff criteria for euthanasia.
[00136] Fig. 11C is a graph showing the change in clinical status for the mice of Fig. 11 A. Grade 1 is pilorection and/or mild ruffled fur, grade 2 is mild hunched posture or mild ruffled fur and reduced activity, grade 3 is hunched posture and mild ruffled fur and reduced activity, grade 4 is hunched posture and ruffled fur and inactive;.
[00137] Fig. 11 D is a Kaplan-Meier curve showing survival of mice receiving the indicated viral doses and combinations.
[00138] Fig. 12A is a schematic depiction of the location of the Nsp12(A19) deletion in the RNA-bound RNA-dependent RNA polymerase (RdRp) complex (PDB 7BV2). The deletion [575-594; shown in black] extends from the end of the fingers region (pink) to the beginning of the palm domain (green) and is predicted to impair binding to RNA. [00139] Fig. 12B is a gel showing wild-type (WT) RdRp complexes (Nsp7, Nsp8, and Nsp12) or mutant complexes containing Nsp12 (SNN, i.e. motif C mutant: SDD to SNN substitution) subunit or Nsp12 (A19) were expressed and purified from Baculovirus infected Sf-9 cells. These were used to assemble in vitro RNA synthesis reactions. Briefly, a 4-mer primer (5’pACGC3’) was extended with 0.1 M ATP, CTP, UTP, in the presence of 0.1 M [a-32P]GTP. Reaction products obtained in the presence or absence of WT RdRp complex were resolved on a denaturing 20% polyacrylamide gel. The Nsp12 (SNN) and Nsp12 (A19)RdRp complexes were significantly impaired for RNA synthesis (compare lanes 13-18 and 19-24 to lane 1 ).
DETAILED DESCRIPTION
[00140] The present disclosure is based on the surprising discovery that polypeptides based on defective SARS-CoV-2 genomes are capable of squelching parental virus replication in vitro and in vivo. Indeed, nucleic acid molecules encoding defective viral genomes (DVGs) that are unable to replicate and/or be packaged on their own, but which still comprise replication and packaging signals, can function as defective interfering particles (DIPs). DIPs compete with parental genomes for limited resources during replication and packaging, thereby leading to reduced parental virus titers. As such, polypeptides based on SARS-CoV-2 defective genomes or nucleic acid molecules encoding such a polypeptide, are potential anti-coronavirus biologic drugs.
[00141] In the Examples below, naturally-occurring SARS-CoV-2 DVGs have been identified, and genetically and functionally characterized. All of the abundant SARS-Cov-2 DVGs isolated had: (i) retained 5’ end sequences, (ii) an in-frame fusion between Nsp1 and Nsp10 (the “Nsp1-10 fusion”), (iii) had retained a frameshift site and the Nsp12 ORF, and (iv) retained 3’ end sequences. It was demonstrated herein that the most abundant DVGs isolated can function as DI particles, genetically interfering with parental SARS-CoV-2 virus replication in vitro and in vivo. It was further noted that the Nsp1-10 fusion product lacks the Nsp1 C-terminal sequences critical for interacting with the mRNA entry channel of the 40s ribosome and inhibiting host translation initiation during viral infection. Therefore, recombinant Nsp1-10 fusion proteins was constructed and showed that, unlike recombinant Nsp1 , the recombinant Nsp1-10 fusion protein was incapable of inhibiting cap-dependent translation when transfected into cells. It was further demonstrated that, upon infection with SARS-CoV-2, cells stably expressing Nsp1-10 exhibited viral yields that were reduced 25- fold compared to controls. In sum, this indicated that the Nsp1-10 exerts a dominant inhibitory effect on parental SARS-CoV-2 replication. [00142] The present disclosure therefore relates to polypeptides capable of limiting the replication of coronavirus and/ or nucleic acid molecules encoding polypeptides capable of limiting the replication of coronavirus, including those comprising a Nsp1-10 fusion, and can provide an effective therapeutic treatment for coronaviruses.
Abbreviations :
[00143] DIP: defective interfering particle
[00144] DVG: defective viral genome
[00145] IFN: interferon
[00146] MOI: multiplicity of infection
[00147] NSP: non-structural protein
[00148] ORF: open reading frame
[00149] PFU: particle forming units
[00150] TIP: therapeutic interfering particle
[00151] According to one aspect, the present application provides an isolated polypeptide capable of limiting the replication of a coronavirus. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus. In some embodiments, the isolated polypeptide comprises a fusion of two polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of three polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of four polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of five polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of six polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of seven polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of eight polypeptides encoded by Nsp1, Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of nine polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of ten polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of 11 polypeptides encoded by Nsp1 , Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 and/or Nsp11. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of ORF1a of a coronavirus and further comprises at least one polypeptide encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least two polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least three polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least four polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least five polypeptides encoded by Nsp12, Nsp13, Nsp14, Nsp15 and/or Nsp16. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises the Nsp12 polypeptide, which includes the -1 ribosomal frameshift site. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one coronavirus structural protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprise at least one of the S protein, E protein, M protein and N protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least two of the S protein, E protein, M protein and/or N protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least three of the S protein, E protein, M protein and/or N protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises each of the S protein, E protein, M protein and/or N protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one coronavirus accessory protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further at least one of the accessory proteins 3a, 6, 7ab and 8. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further at least two of the accessory proteins 3a, 6, 7ab and 8. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further at least three of the accessory proteins 3a, 6, 7ab and 8. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further each of the accessory proteins 3a, 6, 7ab and 8. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b and at least one structural protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b and at least one accessory protein. In some embodiments, the isolated polypeptide comprises a fusion of at least two polypeptides encoded by the viral open reading frames (ORFs) of a ORF1a of a coronavirus and further comprises at least one polypeptide encoded by ORF1 b, at least one structural protein and at least one accessory protein. The terms “fusion” or “fusion polypeptide” as used herein refer to a polypeptide created by the joining of two or more distinct polypeptides or proteins encoded by separate genes. The fusion polypeptide may be generated recombinantly through the genetic engineering of two or more separate ORFs of a ORF1a of a coronavirus into a single fusion gene. The recombinant fusion polypeptide may further comprise linker genes to provide spacing between the different polypeptide components. The fusion polypeptide may also be generated by linking two or more separate polypeptides or proteins together covalently, whether by peptide bonds or other types of chemical linkage (e.g. disulfide bonds) known in the art. The fusion polypeptide may also be generated recombinantly from a naturally-occurring fusion gene. The term “replication” as used herein refers to the action or process of reproducing or duplicating viral RNA and the associated viral proteins by the host cell and includes within this definition the assembly of infective progeny virus particles. As used herein, a polypeptide or nucleic acid is capable of “limiting the replication of a virus” when it reduces the amount of viral RNA and/or associated viral proteins by the host cell by at least half a log compared to a control. The control may comprise an untreated host cell infected with parental virus. The control may also comprise a host cell that is infected with parental virus and treated with a polypeptide encoding a reporter gene or a nucleic acid molecule encoding a polypeptide encoding a reporter gene, wherein expression of the reporter gene has not effect on the replication of the parental virus (e.g. luciferase). Viral replication can be quantified using different standard techniques known in the art (e.g. plaque assay, quantitative real time polymerase chain reaction (qRT-PCR)).
[00152] In specific embodiments, at least two polypeptides fused comprise the coronavirus proteins Nsp1 (variants or fragments thereof) and Nsp10 (variants or fragments thereof), and the fusion polypeptide comprises a Nsp1 moiety and a Nsp10 moiety. The term “moiety” as used herein refers to either a fragment of a polypeptide or the entire polypeptide. As used herein, a “variant” of a protein/polypeptide refers to a full length version of the protein/polypeptide, from any coronavirus, that comprises at least one amino acid difference when compared to the version of said protein/polypeptide normally found in the wild-type coronavirus. The term “fragment” of a protein/polypeptide, as used herein, refers to a protein/polypeptide derived from, but shorter in length than, a full-length protein/polypeptide. The fragment retains the function of the peptide it is a fragment of. The Nsp1 and Nsp10 polypeptides may be derived from wild-type SARS-CoV-2 Nsp1 and Nsp10 genes, respectively, or from variants of said genes, whether naturally occurring or engineered. The isolated polypeptide may also comprise a fusion of Nsp1 and/or Nsp10 moieties derived from other coronaviruses including non-SARS-CoV-2 beta coronaviruses (e.g. SARS-CoV, MERS- CoV, etc.) or alpha coronaviruses (HuCoV-229E, HuCoV-NL63). In some embodiments, the isolated polypeptide comprises a Nsp1 moiety that comprises amino acid residues 1 to 162 of the SARS-Cov-2 wild-type Nsp1 (such as, for example, residues 1 to 162 of SEQ ID NO: 1 ). In some embodiments, the Nsp1 moiety can comprise the amino acid sequence of SEQ ID NO: 3, be a variant of the amino acid sequence of SEQ ID NO: 3 or be a fragment of the amino acid sequence of SEQ ID NO: 3. In additional embodiments, the Nsp1 moiety can be a variant of the amino acid sequence of SEQ ID NO: 1 or a fragment of the amino acid sequence of SEQ ID NO: 1. Variants of the Nsp1 moiety have, in some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1 or 3 and exhibits similar biological activity when compared to the wild-type amino acid sequence of SEQ ID NO: 1 or 3 . The term “percent (%) identity,” as known in the art, is a relationship between two or more polypeptide sequences, as determined by comparing the sequences. The level of identity between two or more sequences can be determined conventionally using known bioinformatics programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991 ).
[00153] In some embodiments, the isolated polypeptide comprises a Nsp10 moiety that comprises amino acid residues 97 to 139 of the SARS-CoV-2 wild-type Nsp10 polypeptide (such as, for example, residues 97 to 139 of SEQ ID NO:2). In some embodiments, the Nsp10 moiety comprises the amino acid sequence of SEQ ID NO:4, be a variant of the amino acid sequence of SEQ ID NO:4, or be a fragment of the amino acid sequence of SEQ ID NO: 4. In additional embodiments, the Nsp10 moiety can be a variant of the amino acid sequence of SEQ ID NO:2 or a fragment of the amino acid sequence of SEQ ID NO: 2. Variants of the Nsp10 moiety have, in some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID 2 or 4 and exhibits similar biological activity when compared to the wild-type amino acid sequence of SEQ ID NO: 1 or 3. In some embodiments, the isolated polypeptide comprises a fusion of Nsp1i-162 (such as, for example residues 1 - 162 of SEQ ID NO: 1 or SEQ ID NO:3) and Nsp1097-i39 (such as, for example residues 97 - 139 of SEQ ID NO: 2 or SEQ ID NO:4) from SARS-CoV-2. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO:5, be a variant of the amino acid sequence of SEQ ID NO:5, or be a fragment of the amino acid sequence of SEQ ID NO:5. Variants of the fusion of Nsp1i-i62 and Nsp1097-i39 have, in some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 , 2, 3, 4 and/or 5 and exhibits similar biological activity when compared to the wild-type amino acid sequence of SEQ ID NO: 1 , 2, 3, 4, and/or 5 (Table 1 ).
Table 1. Sequences for SEQ ID Nos: 1-5
Figure imgf000025_0001
Figure imgf000026_0001
[00154] According to a second aspect, the present application also relates to an isolated nucleic acid molecule coding for the isolated polypeptide described herein. Broadly, this nucleic acid molecule comprises sequential sequences encoding for the at least two polypeptides from the ORF1a of a coronavirus that are operably linked in the same open reading frame and is capable of expressing the isolated polypeptide described herein. In some embodiments, the isolated nucleic acid molecule comprises one or more ribonucleic acid (RNA) residues. In some embodiments, the isolated nucleic acid molecule is a messenger RNA molecule (mRNA). In other embodiments, the isolated nucleic acid molecule comprises one or more deoxyribonucleic acid (DNA) residues. In yet other embodiments, the isolated nucleic acid molecule is a DNA molecule. In some embodiments, the isolated nucleic acid molecule further comprises (a) heterologous gene(s). Examples of suitable heterologous genes include genes that can help boost and antiviral response such as “kill-switches”, interferons (IFNs), short hairpin RNA (shRNA) and/or Cas9. An inducible promoter may be used to drive expression of the heterologous gene.
[00155] According to a third aspect, the present disclosure provides an isolated nucleic acid molecule comprising a defective viral genome (DVG) encoding a therapeutic interfering particle (TIP). As used herein, a “DVG” comprises a coronavirus genome that, due to the emergence of one or more mutation, is lacking essential cis-acting elements and/or is unable to replicate on its own. The DVGs result from errors during replication that led to complementary ends, deleterious point mutations, deletions, insertions, mosaic rearrangements, or any combination of these. As described in the examples below, coronavirus DVGs may be obtained by growing the virus in host cells under appropriate conditions (e.g. a high multiplicity of infection for 15 - 30 passages). As defined herein, a “TIP” comprises a DVG that has lost a critical portion normally required for the ability of the virus to replicate and/or be packaged, but has retained replication and packaging signals that allow it to be replicated and be packaged in the presence of a complete functional virus genome capable of providing any missing functions. In some embodiments, the TIP is capable of limiting the replication of a human coronavirus. The ability of the TIP to limit replication of a human coronavirus can be determined by comparing the viral titers obtained from host cells infected with a human coronavirus and the TIP, compared to control cells that are only infected with a human coronavirus. Viral titers can be measured using standard techniques known in the art. In some embodiments, the TIP is replication defective and can be replicated in the presence of a helper virus. The term “helper virus” as used herein refer to a virus that allows an otherwise-deficient co-infecting virus to replicate, by providing the functions that they have lost. Standard techniques known in the art can be used to assess whether or not a TIP is replication defective on its own in host cells and whether it can become replication competent in the presence of a helper virus. In some embodiments, the TIP is defective for packaging and can be packaged in the presence of the helper virus. Standard techniques known in the art can be used to assess whether or not a TIP is defective in packaging on its own in host cells and whether it can become capable of being packaged in the presence of a helper virus. In some embodiments, the TIP is capable of being enriched upon a plurality of passages in a cell infected with the coronavirus and/or the helper virus at a multiplicity of infection equal to or greater than 1 . As used herein, the terms “passage,” “passaging” or “serial passaging” refers to the in vitro or in vivo process of growing a coronavirus in iterations whereby the virus is allowed to grow for a certain amount of time in a given environment before part of it will be transferred to a new environment and allowed to grow for the same period. This process will be repeated as many times as desired. Enrichment of the TIP following a plurality of passages in a cell infected with coronavirus and/or the helper virus at a multiplicity of infection equal to or greater than 1 PFU per cell can be determined using standard techniques known in the art such as, for example, qRT-PCR. In some embodiments, the TIP encodes the isolated polypeptide defined herein and/or comprises the isolated nucleic acid molecule defined herein. In some embodiments, the human coronavirus is the helper virus that provides the functions needed to allow for the TIP to replicate and/or be packaged. In some embodiments, the human coronavirus and/or the helper virus is from the alpha coronavirus genus. In some embodiments, the human coronavirus and/or the helper virus are 229E or NL63. In other embodiments, the human coronavirus and/or helper virus is from the beta coronavirus genus. In yet other embodiments, the coronavirus and/or helper are 0043, HKU1 , SARS-CoV, MERS-CoV, or SARS-CoV2. In other embodiments, the human coronavirus and/or helper virus is from the gamma coronavirus genus. In yet other embodiments, the human coronavirus and/or helper virus is from the delta coronavirus genus.
[00156] In some embodiments, the isolated nucleic acid molecule that comprises the DVG encoding a TIP comprises, when compared to the nucleic acid sequence of the genome of the coronavirus and/or the helper virus, a first deletion in a ORF1a. In some embodiments, the first deletion starts at a position corresponding to position 749 of GeneBank accession number NC_045512. In some embodiments, the first deletion ends at a first end position corresponding to position 13311 of GenBank accession number NC_044512. In some embodiments, the first deletion encompasses the nucleic acid sequence encoding at least one of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2. In some embodiments, the first deletion encompasses Nsp3. In some embodiments, the first deletion encompasses Nsp4. In some embodiments, the first deletion encompasses Nsp5. In some embodiments, the first deletion encompasses Nsp6. In some embodiments, the first deletion encompasses Nsp7. In some embodiments, the first deletion encompasses Nsp8. In some embodiments, the first deletion encompasses Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least two of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2 and Nsp3. In some embodiments, the first deletion encompasses Nsp3 and Nsp4. In some embodiments, the first deletion encompasses Nsp4 and Nsp5. In some embodiments, the first deletion encompasses Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp6 and Nsp7. In some embodiments, the first deletion encompasses Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp8 and Nsp 9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least three of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3 and Nsp4. In some embodiment, the first deletion encompasses Nsp3, Nsp4 and Nsp5. In some embodiments, the first deletion encompasses Nsp4, Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp5, Nsp6 and Nsp7. In some embodiments, the first deletion encompasses Nsp6, Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp7, Nsp8 and Nsp9 In some embodiments, the first deletion encompasses the nucleic acid sequence of at least four of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3, Nsp4 and Nsp5. In some embodiments, the first deletion encompasses Nsp3, Nsp4, Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp4, Nsp5, Nsp6 and Nsp7. In some embodiments, the first deletion encompasses Nsp5, Nsp6, Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp6, Nsp7, Nsp8 and Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least five of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3, Nsp4, Nsp5 and Nsp6. In some embodiments, the first deletion encompasses Nsp3, Nsp4, Nsp5, Nsp6 and Nsp7. In some embodiments, the first deletion encompasses Nsp4, Nsp5, Nsp6, Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp5, Nsp6, Nsp7, Nsp8 and Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least six of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses Nsp2, Nsp3, Nsp4, Nsp5, Nsp6 and Nsp7. In some embodiments, the first deletion encompasses Nsp3, Nsp4, Nsp5, Nsp6, Nsp7 and Nsp8. In some embodiments, the first deletion encompasses Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 and Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of at least seven of Nsp2, Nsp3, Nsp4,
Nsp5, Nsp6, Nsp7, Nsp8 or Nsp9. In some embodiments, the first deletion encompasses the nucleic acid sequence of each of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8 and Nsp9. As noted above, the TIP described herein must be capable of limiting replication of a human coronavirus.
[00157] In some embodiments, the TIP is capable of limiting the titers of a human coronavirus and/or helper virus by at least one log or more. As used herein, the “titer” of a virus refers to the concentration of infectious viral particles. Infectious viral particle titers may be quantified using techniques known in the art such as, for example, plaque formation assays, focus formation assays, end point dilution assays and protein- based virus quantification assays. In some embodiments, the DVG encoding a TIP has the nucleic sequence of GI.285 (SEQ ID NO:6), GI.249 (SEQ ID NO:7), GI.616 (SEQ ID NO:8), GI.50 (SEQ ID NO:9), GI.55 (SEQ ID NO:10) or GI. 535 (SEQ ID NO:11 ) or a corresponding mRNA version of these nucleic acid sequences (Table 2). DVGs with nucleic sequences that comprise intact replication and packaging sequences, such as the aforementioned DVG sequences, are capable of replicating and expanding in the presence of wild-type and/or helper viruses. In some embodiments, the DVGs can include additional heterologous genes, such as, for example, “kill-switches”, interferons (IFNs), inducible Caspase triggers, short hairpin RNA (shRNA) and/or Cas9. In some embodiments, the DVGs can be delivered as a therapeutic interfering particle. In additional embodiments, the DVGs can be delivered as a nucleic acid vector to be expressed in coronavirus-infected cells. Such DVGs may be prepared in vitro by genetic engineering using standard techniques and delivered as a therapeutic. The nucleic sequence of the DVG can be determined using standard sequencing techniques known in the art.
Table 2. Sequences for SEQ ID Nos: 6-11
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
[00158] According to a fourth aspect, the present disclosure provides a TIP comprising the isolated poypeptide described herein and/or the isolated nucleic acid molecule described herein. The TIP comprising the isolated polypeptide and/or the isolated nucleic acid may be generated by repeated serial passaging of a parental coronavirus, at high multiplicities of infection (MOI) in a suitable cell line. The term “MOI” as used herein refers to the number of virions added per cell during infection. A high MOI, for the purpose of generating DVGs and TIPs, is known in the art and includes, but is not limited to, MOIs of 1 - 10 particle forming units (PFU) per cell. For serial passaging of coronavirus at high MOI, cells lines that have decreased anti-viral responses (e.g. IFN responses) are preferred. Examples of cells lines that may be suitable for the repeated serial passaging of coronavirus at a high MOI include, but are not limited to, Vero E6, Vero81 , Vero SLAM, MA104, BGM, Caco-2, Calu-3 and Huh7.5. In some embodiments, the TIP comprising the isolated polypeptide and/or the isolated nucleic acid may be generated by serially passaging the parental virus at an MOI=3 pfu/cell. As described herein, DVGs and TIPs were abundant after 14 - 30 passages in Vero E6 cells at an MOI=3 pfu/cell. The TIP comprising the isolated polypeptide and/or the isolated nucleic acid may be generated by co-transfecting cells with a synthetic recombinant DVG genome and the genome of a helper virus. Similarly, cells lines that stably express synthetic recombinant DVG genome and the genome of a helper virus can be used to generate the TIP comprising the isolated polypeptide and/or the isolated nucleic acid. The synthetic recombinant DVG genome may be prepared using standard techniques known in the art. In some cases, the TIP comprising the isolated isolated polypeptide and/or the isolated nucleic acid may comprise an engineered virus-like particle (VLP) that encapsulates the isolated nucleic acid and iscapable of delivering it into a recipient cell infected with coronavirus. Techniques for the preparation of VLPs and the delivery of proteins to cells via VLPs are known in the art.
[00159] According to a fifth aspect, the present disclosure provides a pharmaceutical composition comprising: (i) the isolated polypeptide derived herein and/or the isolated nucleic acid defined herein, and (ii) one or more pharmaceutically acceptable excipient. The term “pharmaceutically acceptable excipient” is known in the art and includes, but is not limited to, 0.01 - 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable excipients may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. In some embodiments, the pharmaceutical composition is formulated for nasal administration. Other suitable routes of administration for the pharmaceutical composition includes nasal, intranasal, oral, intravenous, intramuscular, subcutaneous, rectal and inhaled. [00160] According to a sixth aspect, the present disclosure provides a method of treating a coronavirus infection in a subject in need thereof, whereby the method comprises administering a therapeutically effective amount of the isolated polypeptides defined herein, the isolated nucleic acid defined herein and/or the pharmaceutical composition defined herein to the subject so as to reduce the replication of the coronavirus. As defined herein, “therapeutically effective amount” refers to a quantity of the isolated polypeptide described herein, the isolated nucleic acid described herein and/or the pharmaceutical composition defined herein that is effective in mitigating at reducing replication of the coronavirus and/or mitigating one or more symptom of coronavirus infection when administered to an individual in need thereof. It is also understood herein that a therapeutically effective amount of the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition may be provided in different dosage forms and by different routes, both alone or in combination with other therapeutic agents indicated for the treatment of human coronavirus infections (e.g. remdesivir, sotrovimab, dexamethasone). In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided intra-nasally to the subject in need thereof. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided by inhalation. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided by injection. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided orally. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to the subject in need thereof in one or more doses. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof after the presence of a coronavirus is detected in the subject. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof before the presence of a coronavirus is detected in the subject. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof before and after the presence of a coronavirus has been detected in the subject.
[00161] In some embodiments, the method of treating a coronavirus infection in a subject in need thereof comprises prophylactically administering a therapeutically effective amount of the isolated polypeptides defined herein. As used herein, “prophylactically administering” refers to administering a therapeutically effective amount of the isolated polypeptide, the isolated nucleic acid and/or the pharmaceutical composition defined herein to a subject before they are exposed to and/or infected by coronavirus, including but not limited to before the presence of coronavirus is detected in the subject. It is also understood herein that “prophylactically administering” a therapeutically effective amount can also refer to administering a therapeutically effective amount of the isolated polypeptide, the isolated nucleic acid and/or the pharmaceutical composition defined herein to a subject after exposure to and/or infection by coronavirus, including but not limited to after the presence of coronavirus has been detected in the subject, but before the development of symptoms of coronavirus infection. In some embodiments, the isolated polypeptide, isolated nucleic acid and/or pharmaceutical composition is provided to a subject in need thereof prophy lactically and in combination with other prophylactic agents. In some embodiments, the subject in need thereof is a mammal. In some embodiments, the subject in need thereof is a human being. In some embodiments, the subject in need thereof is a child. In some embodiments, the subject in need thereof is an adult.
[00162] The present disclosure thus provides polypeptides capable of limiting the replication of coronavirus and/ or nucleic acid molecules encoding polypeptides capable of limiting the replication of coronavirus, including those comprising a Nsp1-10 fusion and/or Nsp12.
EXAMPLE 1 - Material and methods
[00163] The present example provides the material and methods used in subsequent examples.
[00164] Virus and cells: Vero E6, HEK 293T, and HEK 293T-ACE2 (PMID 34110264) cells obtained from the American Type Culture Collection (Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 100 U/ml penicillin and streptomycin, 1 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) (Gibco), 2 mM glutamine (Gibco), and 10% heat-inactivated fetal bovine serum (FBS) at 37°C in 5% CO2. SARS-CoV-2 (hCoV-19/Canada/CN-VIDC-01/2020; GISAID accession no. EPI_ISL_425177) was a donation. In order to obtain HEK 293T-ACE2 cells stably expressing Nsp1-10, HEK 293T-ACE2 cells were transfected by calcium phosphate with pcDNA3.1- 3xFLAG-Nsp1/10 (5 pg). Expression of Nsp1-10 was verified by Western blotting and probing with a monoclonal anti-FLAG antibody (1 :1000 dilution) produced in mouse (Sigma- Aldrich, #F1804).
[00165] Generation of defective interfering particles: Vero E6 cells (106 cells/well) grown in six-well plates were infected with SARS-CoV-2 at MOI of 3 for 60 min. The virus was removed and replaced with 2 mL of fresh complete DMEM media. Cells were monitored daily, and viral supernatant was harvested after the appearance of cytopathogenic effect (CPE) between 24 and 48 h. Fifty percent of the supernatant containing passage 1 (P1 ) of the virus was used to infect a new batch of Vero E6 cells, and the remaining R1 virus was frozen as two equal-volume aliquots at -80 °C. After 1 h of infection, the virus was removed and replaced with 2 ml_ fresh complete DMEM media. Cells were monitored daily, and the supernatant was harvested after the appearance of CPE between 24 and 48 h. This was passage 2 (P2) of the virus. The same procedure was repeated until passage 30 (P30) was reached. Total cellular RNA at each passage was extracted and stored at -80 °C for future use. The same viral seed stock was used in the Examples further below.
[00166] Antibodies: Antibodies used in the examples were: anti-Nsp1 (GeneTex, GTX135612), anti-Nsp10 (Pro-Sci Inc, #9179), anti-RPS6 (CST, #2317 ), anti-FLAG (Sigma- Aldrich, #F1804), anti-RPL7 (Novus Biologicals, #NB100-2268, anti-GAPDH (Abeam, #ab8245), anti- -actin (Abeam, #ab8226), anti-eEF2 (CST, #2332), and antihnRNPAI (CST, #8443).
[00167] Plasmid construction: To generate pHiC IntIK G1.616, the EMCV-renilla luciferase sequence released from pHiC IntU G1.616 by restriction enzyme digestion using Alel and Stul and was replaced with a G block non-directionally containing Nsp13-N sequence bounded by Alel and Stul restriction sites. pHiC IntI K G1.50 was generated in the same manner as pHiC IntI K G1.616, but starting with the pHiC IntU G1.50 vector, instead of pHiC IntU G1.616. pHiC IntI K G1.55 was cloned in two steps. In the first step, a G block consisting of the SARS-CoV-2 5’ UTR, Nsp1-10 fusion protein, Nsp11, and Nsp12 N- terminus bounded by Sphl and Sacl restriction sites was directionally cloned into pHiC IntU G1.50 to give pHiC IntU G1.55. In the second step, the EMCV-renilla was released from pHiC IntU G1.55 by restriction enzyme digestion using Sacl and EcoRI and a G block that has Sacl and EcoRI restriction sites and contains the Nsp12 C-terminus, Nsp13-N fusion sequence, and ORF10 (aa 1-21 ) was then cloned directionally into pHiC IntU G1.55. pcDNA3.1_Nsp1-3xFLAG containing a Nhel site downstream of the CMV promoter and a BamHI site upstream of the BGH pA signal was a donation. The Nsp1-3xFLAG insert was removed by restriction digest with Nhel/BamHI. A G block containing the Nhel/BamHI restriction sites and 3xFLAG-spacer-Nsp1 sequences was directionally cloned to generate pcDNA3.1_3xFLAG-Nsp1. Using a similar strategy, pcDNA3.1_3xFLAG-Nsp1 (K164A/H165A) was generated using a similar G block harboring the two missense mutations. To obtain pcDNA3.1_3xFLAG-Nsp1/10 a two-step cloning was implemented: In the first step, a PCR product was amplified from cDNA obtained from Exp#1 P30 as template using the primers Narl-f (5’AGTCCACAAGCACGGCGCCGATCTAAAGTCA3’ SEQ ID NO: 12) and Narl/BamHI-r
(5’GATTGTTGTCAATGGCGCCGGATCCCTACTGAAGCATGGGTTCGCGGA3’ SEQ ID NO: 13). Narl/BamHI restriction digest of pcDNA-3.1_3xFLAG-Nsp1 released three fragments. After cutting with Narl, the PGR product was cloned into the linearized backbone and appropriate recombinants selected. In the second step, the BamHI/Narl fragment from pcDNA-3.1_3xFLAG-Nsp1 was directionally sub-cloned into one of the selected recombinants. pET15b-His6-Nsp1 , pET15b-His6-Nsp1/10, and pET15b-His6-Nsp1 (K164A/H165A) were cloned using G blocks with Ndel and BamHI positioned at the ends. All cloned G blocks were Sanger sequenced to ensure the absence of mutations.
Table 3. Nucleotide sequence of constructs used
Figure imgf000041_0001
[00168] In vitro synthesis of capped mRNA: Plasmids carrying DI genomes were linearized by restriction enzyme digestion using BsmBI or Esp3l. DNA was purified by phenol-chloroform extraction, back extracted with H2O, passed through a column of Sephadex™ G-50 Superfine beads (GE Healthcare, #17-0041-01 ), precipitate with 100% ethanol, washed with 70% ethanol, and resuspended in RNase-free H2O at a concentration of 1 pg/pl. Using linearized plasmid as a template, mRNA was synthesized in vitro using the T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320) according to the manufacturer’s instructions. Uncapped mRNA was capped in a one-step reaction using Vaccinia Capping System (NEB, # M2080S) and mRNA Cap 2’-O-Methyltransferase (NEB, #M0366S). RNA clean-up was performed via phenol-chloroform extraction as described above for clean-up of linearized DNA. Concentrations were quantitatively measured by NanoDrop™ 1000 (Thermo Scientific). Cap-1 mRNA was analyzed alongside ssRNA ladder (NEB, # N0362S) on a 1% agarose-formaldehyde denaturing gel to confirm size and quality.
[00169] RNA transfections: VeroE6 cells were seeded in a 24-well plate in DMEM supplemented with 10% FBS (Gibco, #12483-020, 1% penicillin-streptomycin (Wisent, # 450- 200-EL), and 1x non-essential amino acids (Wisent, # 321-011-EL) at a density of 2x105 cells/well. The next day, the medium was changed to 200 pL opti-MEM (Gibco, 31985070). Cells were transfected with Lipofectamine™ 3000 (Invitrogen, #L3000015), and transfection mixes were prepared as recommended by the manufacturer. Essentially, 500 ng of cap-1 mRNA was added to each well of mock- or SARS-CoV-2 infected cells and left to incubate for 22 hours before downstream processing.
[00170] RNA extraction: Total RNA was extracted from viral supernatants using QIAamp™ Viral RNA Mini Kit according to the manufacturer’s instructions (Qiagen, # 52904). Total RNA was extracted from SARS-CoV-2 infected cells using the Nucleospin RNA mini kit for RNA purification according to the manufacturer’s instructions (Macherey-Nagel, #740955.50). For isolation of RNA from DI RNA-transfected, uninfected cells, cells were lysed with 500 pL TRIzol™ directly in the plates and incubated for 5 minutes. Total RNA was extracted according to the manufacturer’s instructions (Invitrogen).
[00171] Northern blot analysis: For all Northern blots, total RNA was quantified using the NanoDrop™ 1000 (Thermo Scientific). RNA from each sample was electrophoresed on a 1.2% formaldehyde-agarose gel. Following electrophoresis, the RNA ladder lane was excised and stained with SYBR™ Gold Nucleic Acid Gel Stain (Invitrogen). Northern blot transfers were performed onto Hybond N+ membrane as previously described using 20 x SSC. Following transfer, the membrane was UV-crosslinked at 1.2 x 105 uJ/cm2 The membrane was pre-hybridized with hybridization buffer (50% formamide, 10% dextran sulfate, 0.8M NaCI, 5x Denhardt’s solution, 50mM Tris 7.5, 0.1% sodium pyrophosphate, 100ug/mL salmon sperm DNA, 0.5% SDS) for 16 hours at 42°C, hybridized with the radioactively probe for 16 hours at 42°C. Washes were performed at 65°C twice for 25 minutes each with 0.1% SDS/2x SSC, 0.1% SDS/1x SSC, and 0.1% SDS/0.5x SSC. Autoradiographs were obtained by exposing the membrane to X-ray film (BioMax™ XAR, Kodak).
[00172] Direct RNA nanopore sequencing. Before sequencing, the extracted total RNA was quantified with the “Qubit RNA high sensitivity” quantification kit (Q32855 ; ThermoFisher Scientific) and its quality was profiled on a “High Sensitivity RNA ScreenTape” (5067-5579 ; Agilent). Only high-quality samples were sequenced. The total RNA was sequenced on a MinlON flow-cell (FLO-MIN106 ; Oxford Nanopore Technologies) using the “Direct RNA sequencing” library preparation kit (SQK-RNA002 ; Oxford Nanopore Technologies). The SQK-RNA002 library preparation protocol was followed (version DRS_9080_v2_revM_14Aug2019) as provided from Oxford Nanopore Technologies (abbreviated as ONT) with the following modifications. The library preparation started with 1 pg of total RNA for the passages 1 and 29 of experiment number #1, 3 pg of total RNA for the passage 15 of experiment number #1 and 2 pg of total RNA for the passages 1 , 14 and 30 of experiment number #2. In cases where the starting material was 1 pg of total RNA, the following protocol was used. The first adaptor of the library preparation kit was ligated on the RNA in a 15 pl solution with the following components: 3 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 1 pg of total RNA, 0.5 pL of Recombinant RNase Inhibitor (stock: 40 Units/ul ; 2313A ; Takara), 1 pL of RT Adapter (RTA ; ONT); 1.5 pL of T4 DNA ligase (stock: 2M U/ml ; M0202 ; New England Biolabs), top up the solution to 15 pL with nuclease-free water. This solution was incubated at room temperature for 20 minutes and subsequently mixed with a 23 pL solution named “reverse transcription master mix” that had the following components: 9 pL of nuclease-free water, 2 pL of 10mM dNTPs (N0447S ; New England Biolabs), 8 pL of 5x Superscript™ IV reverse transcription (SSIV RT) buffer (18090010 ; ThermoFisher Scientific), 4 pL of 0.1 M dithiothreitol (DTT) (18090010 ; ThermoFisher Scientific). Next, 2 pL of SSIV RT (18090010 ; ThermoFisher Scientific) were added and the whole reaction was incubated at 50°C for 2 hours and 50 minutes, then at 70°C for 10 minutes and then the sample was brought to 4°C before proceeding with a 1.8X volume of “RNACIean XP” beads cleanup (A63978 ; Beckman Coulter) and one wash of 150 pL with 70% EtOH. The material was then eluted from the beads with 20 pL of nuclease-free water and the second adaptor was ligated in a 40 pL solution containing the following: 20 pL of reverse-transcribed RNA, 8 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 6 pL of RNA Adapter (RMX ; ONT), 2.5 pL of nuclease-free water, 0.5 pL of Recombinant RNase Inhibitor (stock: 40 Units/ul ; 2313A ; Takara), 3 pL of T4 DNA Ligase (stock: 2M U/ml ; M0202 ; New England Biolabs). The reaction was incubated at room temperature for 20 minutes followed by 1X volume of “RNACIean XP” beads cleanup (A63978 ; Beckman Coulter) and two washes of 150 pL with the Wash Buffer (WSB; ONT). The material was eluted, from the beads, in 21 pL of Elution Buffer and 1 pL of the solution was quantified with the “Qubit 1X dsDNA high sensitivity” kit (Q33230 ; ThermoFisher Scientific). Approximately 200-250 ngs of RNA/cDNA hybrid were recovered. After priming the MinlON flow-cell as per the ONT protocol the following solution was loaded: 20 pL of prepped RNA/cDNA hybrid in Elution Buffer, 17.5 pL of nuclease-free water, 37.5 pL of Rapid Running buffer (RRB) (ONT). The duration of the sequencing run was up to 72 hours or until no pores were available for sequencing. In cases where the starting material was 2 or 3 pg of total RNA, the following protocol was used. The first adaptor of the library preparation kit was ligated on the RNA in a 30 pL solution with the following components: 6 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 2 or 3 pg of total RNA, 1 pL of Recombinant RNase Inhibitor (stock: 40 Units/pL ; 2313A ; Takara), 1 pL of RT Adapter (RTA ; ONT); 3 pL of T4 DNA ligase (stock: 2M U/ml ; M0202 ; New England Biolabs), top up the solution to 30 pL with nuclease-free water. The solution was incubated at room temperature for 20 minutes and subsequently mixed with a 46 pL solution named “reverse transcription master mix” with the following components: 18 pL of nuclease-free water, 4 pL of 10mM dNTPs (N0447S ; New England Biolabs), 16 pL of 5x SSIV RT buffer (18090010 ; ThermoFisher Scientific), 8 pL of 0.1 M DTT (18090010 ; ThermoFisher Scientific). Next, 4 pL of SS IV RT (18090010 ; ThermoFisher Scientific) were added and the whole reaction was incubated at 50°C for 2 hours and 50 minutes, then at 70°C for 10 minutes and the sample was brought to 4°C before proceeding with a 1.8X volume of “RNACIean XP” beads cleanup (A63978 ; Beckman Coulter) and one wash of 300 pL with 70% EtOH. The material was then eluted from the beads with 40 pL of nuclease-free water and the second adaptor was ligated in a 80 pL solution containing the following: 40 pL of reverse-transcribed RNA, 16 pL of NEBNext Quick Ligation Reaction Buffer (stock: 5X ; B6058 ; New England Biolabs), 6 pL of RNA Adapter (RMX ; ONT), 11 pL of nuclease-free water, 1 pL of Recombinant RNase Inhibitor (stock: 40 Units/pL ; 2313A ; Takara), 6 pL of T4 DNA Ligase (stock: 2M U/mL ; M0202 ; New England Biolabs). The reaction was incubated at room temperature for 20 minutes followed by 1X volume of “RNACIean XP” beads cleanup (A63978 ; Beckman Coulter) and two washes of 150 pL with the Wash Buffer (WSB; ONT). The material was eluted, from the beads, in 38.5 pL of Elution Buffer and 1 pL of the solution was quantified with the “Qubit 1X dsDNA high sensitivity” kit (Q33230 ; ThermoFisher Scientific). Approximately 400-750 ngs of RNA/cDNA hybrid were recovered. After priming the MinlON flow-cell as per the ONT protocol we loaded the following solution: 37.5 pL of prepped RNA/cDNA hybrid in Elution Buffer, 37.5 L of RRB buffer (ONT). The duration of the sequencing run was up to 72 hours or until no pores were available for sequencing.
[00173] RNA sequencing data processing: RNA sequencing data for all passages were base called by guppy 3.4.4 (Oxford Nanopore Technologies) using the high-accuracy model. The sequenced reads were first adapter-trimmed using porechop (https://github.com/rrwick/Porechop) and then aligned to the reference sequence database consisted of the SARS-CoV-2 genome (GenBank: NC_045512.2), yeast ENO2 cDNA (SGD: YHR174W), human ribosomal DNA complete repeat unit (GenBank: U13369.1 ), C. sabaeus genome (NCBI Annotation Release 100) utilising minimap2 v2.17 (PMID: 29750242) with options “-x splice -N 32 -un.” Then reads that are mapped on viral genome were extracted from alignment and re-mapped on viral reference (GenBank: NC_045512.2) for further quality improvement using minimap2 with parameters “-k 8 -w 1 -splice -g 30000 -G 30000 - A1 -B2 -02,24 -E1,0 -CO -z 400,200 -no-end-flt -junc-bonus=100 -F 40000 -N 32 -splice- flank=no -max-chain-skip=40 -un -junc-bed=JUNCTIONS -p 0.7”. JUNCTIONS file was downloaded from UCSC Table browser (track ‘Transcriptome Kim’, table ‘Known transcripts’). Chimeric reads, secondary and supplementary alignments were filtered out according to the flags from minimap2 using samtools v1.11 (http://www.htslib.org/, PMID: 19505943) and picard V2.23.8 (https://github.com/broadinstitute/picard). Mapped transcripts were collapsed into transcript models using PINFISH (https://github.com/nanoporetech/pinfish.git), RATTLE
(https://github.com/comprna/RATTLE.git) and TAMA collapse
(https://github.com/GenomeRIK/tama.git). Coverage depth was calculated using bedtools genomecov V2.29.2 (PMID: 25199790, https://github.com/arq5x/bedtools2) and plotted using log-transformation with pseudocount of 1. Transcript model abundance was calculated by mapping viral reads back on transcript models using minimap2 and parsing output .paf files using custom Python 3.7.3 scripts. Only leader-containing transcript models were shown. Plots are made by custom Python scripts (seaborn v0.9.0, matplotlib v3.2.1).
[00174] Long-range PCRs: To prepare full-length cDNA for long-range PCRs, total RNA was reverse transcribed using Superscript™ IV Reverse Transcriptase and a gene-specific primer: (5’CTCCTAAGAAGCTATTAAAATCAC3’ SEQ ID NO: 27) according to the manufacturer’s instructions (Invitrogen, #18090010) that targets the 3’UTR of SARS-CoV-2. Single stranded DNA was obtained by RNase H (NEB, #M0297S) treatment and the resulting cDNA was diluted 10-fold. Long-range PCRs were performed using LA-Taq DNA Polymerase Hot-Start Version (TaKaRa, #RR042B) and 2 pL of diluted cDNA as template. Cycling conditions were implemented as recommended by the manufacturer. Primers are indicated in
Table 4. Long-range PCR products were sequenced by Sanger sequencing.
Table 4: Primer sequences
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
[00175] RT-qPCR: Complementary DNA was generated either with M-MuLV reverse transcriptase (NEB, M0253L) or Superscript™ IV VILO™ Mastermix (ThermoFisher, #11756050) using random hexamer primers. The cDNA was diluted 10-fold and used as template for qPCR using SsoFast™ Evagreen™ Supermix (Bio-Rad, #1725201 ). Cycling conditions consisted of an initial denaturation of 95°C/30 seconds followed by 98°C/5 seconds, 60°C/5 seconds (39 cycles), and 65°C to 95°C incremented at a rate of 0.5°C/min. for melt curve acquisition. Primer pairs used are listed in Table 4.
[00176] Polysome Fractionation: HEK293T cells were seeded in 10 cm dishes at 5x10s cells/well in DMEM supplemented with 10% BGSS, 1% penicillin/streptomycin, and 1% L- glutamine (Wisent). The next day, cells were transfected by calcium phosphate using 10 pg of each plasmid. Cells were washed and fresh medium re-applied 6-8 hours posttransfection. Twenty-four hours post-transfection, cells were harvested in ice cold PBS containing 100 pg/ml cycloheximide. Cells were pelleted at 4°C and lysed in 425 pL hypotonic lysis buffer (5 mM Tris-CI (pH 7.5), 2.5 mM MgCI2, 1.5mM KCI). Cycloheximide (100 pg/ml), DTT (2 mM), Triton X-100 (0.5%) and sodium deoxycholate (0.5%) were each added to a final indicated concentration and the samples briefly vortexed. Lysates were cleared by centrifugation at 16,000 x g for 2 min at 4°C. Lysate (400 pl) was layered onto a 10-50% sucrose gradient. The gradients were centrifuged at 217,290 xg for 2 h at 4°C in an SW40 Beckman rotor. Fractions were collected using the Teledyne ISCO Foxy R1 collector while monitoring the UV 254 profile. Proteins were precipitated from each fraction with 10% trichloroacetic acid and collected by centrifugation at 16,000 x g for 30 min 4°C. The pellet was washed with 500 pL acetone, centrifuged at 4°C for 10 min. at 16,000 x g, and dried under vacuum (Eppendorf Vacufuge). Protein pellets were resuspended in 1x SDS sample buffer and analyzed on a 10% SDS-PAGE gel. Resolved proteins were transferred at 4°C onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad™) and probed by immunoblotting.
[00177] Measurement of protein synthesis in vitro and in cellula-. For in vitro translations, a FF/EMCV/Ren bicistronic reporter mRNA previously described was in vitro transcribed using SP6 RNA Polymerase (NEB, #M0207S) and co-transcriptionally capped with m7GpppG RNA Cap Analog (NEB, #S1404S). Nuclease-treated rabbit reticulocyte lysate (RRL) (Promega, #L4960) was programmed with 20 ng/pl mRNA and recombinant proteins. Recombinant proteins were pre-incubated reaction components for 5 min at 30°C before addition of the mRNA. After 1 hour at 30°C, reactions were placed on ice and 10 pg/ml cycloheximide was added to stop the reaction. Luciferase activity was measured on a Berthold™ Lumat LB 9507 luminometer. For [35S]-methionine/cysteine labeling, HEK 293T cells were seeded in a 6-well plate at a density of 1x10s cells/well in DMEM supplemented with 10% BGSS, 1% penicillin-streptomycin, and 1% L-glutamine. Cells were transfected with the indicated pcDNA3.1 expression plasmids and 6-8 hours post-transfection, washed three times with PBS, trypsinized, and seeded into a 24-well plate in technical duplicates. Twenty- four hours post-transfection, medium was exchanged for 45 min with methionine-free, cysteine-free DMEM (Gibco, #21013-024) supplemented with 10% FBS, after which 22 uCi of 35S-Methionine/Cysteine Protein Labeling Mix (Perkin Elmer, #NEG772007MC) was added per well. Labelling was performed for 15 min at 37°C/5% CO2 after which cells were lysed in RIPA buffer (20mM Tris-CI 8.0, 100mM NaCI, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.01 mg/ml aprotinin, 0.002 mg/ml leupeptin, 2.5uM pepstatin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). Lysates were spotted onto 3MM Whatman paper and the proteins precipitated with 10% trichloroacetic acid (TCA). TCA insoluble radiolabeled proteins were quantified by scintillation counting, and CPMs were normalized to the total protein amounts determined for each sample by the DC Protein Assay (Bio-Rad, #5000112). Radiolabeled lysates were also resolved by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad™) when Western blots had to be performed.
[00178] Recombinant protein purification: Recombinant His6-tagged Nsp1 , Nsp1 (K164A/H165A) and Nsp1/10 proteins were purified BL21 (DE3) cells expressing pET15b- based expression vectors. Single colonies were picked and 20 ml_ cultures were grown overnight in LB media supplemented with ampicillin (100ug/ml). Cultures were used to inoculate 1 L of LB/amp (100 pg/ml) and induced with isopropylthio-p-galactoside (IPTG) (0.5 mM) when the OD600 reached 0.8, at which point cultures were moved to 18°C for 16 hours. Cells were pelleted by centrifugation at 5000rpm for 20 min, resuspended in 20 mL Nsp1 sonication (50mM HEPES-KOH, 500mM KCI, 5mM MgCI2, 40mM imidazole, 10% glycerol, 1 mM PMSF, 0.01 mg/ml aprotinin, 0.002 mg/ml leupeptin, 2.5uM pepstatin, 0.5mM dithiothreitol, pH 7.6), and lysed by sonication(Heat systems ultrasonics; 10 pulses at 1 pulse/sec). The lysate was cleared by centrifugation at 4°C for 48 minutes at 48,000 x g. Proteins were purified on Ni-NTA agarose beads (Qiagen, #30210), washed twice with Nsp1 sonication buffer, and eluted with Nsp1 elution buffer (50mM HEPES-KOH, 500mM KCI, 5mM MgCI2, 300mM imidazole, 10% glycerol, pH 7.6). Eluted protein fractions were dialyzed overnight at 4°C in Nsp1 dialysis buffer (40mM HEPES-KOH, 200mM KCI, 4mM MgCI2„ 10% glycerol, 1 mM dithiothreitol, pH 7.6). Protease inhibitors and dithiothreitol were always freshly added.
[00179] In vivo murine infections: K18-ACE2-overexpressing mice (Stock No: 034860) were obtained from the Jackson Laboratory (Bar Harbor, Maine) and maintained by back- crossing to strain C57BL/6J (Stock No. 000664). Mice were infected with 104 PFU intranasally under ketamine-xylazine anaesthesia. Body weight and clinical score was monitored at least daily and twice at peak seakness days (days 5-7 post infection). Clinical signs were given a value 0 to 4 according to the scale: 0, normal appearance and behavior; 1 , hunched but active; 2, hunched and milde ruffled and less activity; 3, moderate hunched posture, ruffled fur, less active, shallow breathing. Mice were humanely sacrificed upon reaching 20% weight-loss or a clinical score of 3.
[00180] Plaque assays. Vero E6 cells were plated in 24-well plates (105 cells/well) and incubated overnight at 37 °C. Virus-containing media was serially diluted (10“1-10“6) with DMEM media into 96-well plates. Then, 100 L of each dilution was added in duplicate to Vero E6 cells in the 24-well plates, and samples were incubated at 37 °C in 5% CO2 for 1 h with rocking every 15 min. After 1 h incubation, the virus-containing media was removed and 1 mL of pre-warmed plaquing media (MEM media containing 2% FBS and 0.75% methylcellulose) was added to each well. Plates were incubated at 37 °C in 5% CO2 for 3 days to allow plaque formation. On day 3, methylcellulose overlays were gently removed, and cells were fixed by adding 1 mL of 4% paraformaldehyde in PBS to each well. After incubation at room temperature for 30 min, the fixative was removed, plates were washed with dH2O, and 1 mL of 1% (w/v) crystal violet in 20% methanol was added to each well. The crystal violet solution was removed after 30 min, and the plates were washed with water. Plaques were only counted in wells in which there were 5-30 plaques.
[00181] Immunoblotting (Nsp1-10 fusion). Vero E6 cells infected with P2, P15, and P30 of SARS-CoV-2 passage were harvested at 24 h post-infection. Cells were washed three times with PBS before lysing with RIPA buffer (50mM Tris-HCI (pH 7.4), 150mM NaCI, 1% Triton-X-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA) containing fresh protease inhibitors and 1 unit of Benzonase (Millipore; Burlington, MA, USA) per sample. Proteins were denatured at 95 °C for 10 min, separated by SDS-PAGE, and transferred to PVDF membranes for immunoblotting. Imaging was performed using an Odyssey™ CLx Imaging System (LI-COR Biosciences; Lincoln, NE, USA).
[00182] Renilla luciferase assays. Vero E6 cells (105 cells/well) seeded in 24-well plates were infected with SARS-CoV-2 at an MOI = 1 for 1 h and then transfected in duplicate with in vitro synthesized RLuc, GI.616-EMC/RLuc, and GI.616-TRS/RLuc RNA. The supernatant was collected at 24 h post-infection, clarified, and fifty percent of the virus was used in serial infections of Vero E6 cells. At 24 h postinfection of each passage, growth media was removed, cells were washed with PBS, and lysed with 100 pL renilla luciferase assay lysis buffer (Promega, #E2810) for 15 min at room temperature. The lysate was collected and stored at -80 °C until further use. For luciferase assays, 20 pL from each passage was aliquoted into white 96-well microplates (Greiner bio-one) in duplicates for Renilla luciferase activity measurements. One hundred microliters of renilla luciferase assay reagent (Promega, #E2810) was added to each well, and luciferase activity was measured immediately using a Synergy HTX plate reader (Biotek; Winooski, VT, USA). In addition to the virus supernatant used in serial infections, the remaining virus was collected for titering. Additionally, total cellular RNA from each passage of cells was also extracted and stored at -80 °C for future use.
[00183] Determining the relative abundance of DVGs across viral passages. Cellular RNA from P20, P25, and P30 was reverse transcribed according to the manufacturer’s protocol using Superscript™ IV Reverse Transcriptase (Invitrogen, #18090010). The resulting cDNA was treated with RNase H (NEB, #M0297S) and diluted 10-fold. Diluted cDNA was used as a template for LR-PCR using the A1/A2 primer pair and LA-Taq DNA Polymerase Hot-Start Version (TaKaRa, #RR042A). The major long-range product was gel purified and cloned by TA cloning into pGEM-T Easy using the pGEM™-T Easy Vector System (Promega, # A1360) according to the manufacturer’s instructions or by blunt-end ligation into pBluescript II KS (+) using EcoRV. Minipreps were performed by the alkaline lysis method to obtain plasmid DNA from each clone which was then Sanger sequenced.
[00184] Determining the growth rate, packaging efficiency, and transmission efficiency of SARS-CoV-2 and DIPs. Vero E6 cells were infected with SARS-CoV-2 at an MOI of 1 , and DI mRNA was transfected 8 h post-infection. Twenty-two hours posttransfection, the resulting supernatant was serially passaged three times, once every 24 h. Cellular RNA was extracted at the indicated time points postinfection in P2 (4, 8, and 24 h) and at 4 h in P3. RNA was also extracted from P2 supernatant at 24 h. After performing RT- qPCR, the growth rate of SARS-CoV-2 was then calculated by the 2-ACt method using GAPDH as a control. The resulting values were normalized to the 4 h time point. To calculate the percentage of genomes packaged and transmission efficiency, the DI and SARS-CoV-2 RNA copy numbers were determined after RT-qPCR, from a standard curve established using recombinant RNA standards. The percentage of packaged DI or WT virus mRNAs was then calculated as follows: 100 * (mRNA copy number in P2 supernatant)/(mRNA copy number in P2 supernatant + mRNA copy number in P2 cells). The transmission percentage was calculated as follows: 100 * (mRNA copy number in P3 cells at 4 hr)/(mRNA copy number in P2 cells at 24 h).
[00185] Differential detergent fractionation. HEK-293T cells were seeded in a six-well plate at a density of 106 cells/well. In each well, cells were transfected by calcium phosphate using 5 pg empty pcDNA3.1 or pcDNA3.1 expressing the indicated proteins. Twenty-four hours post-transfection, cells were scraped in cold PBS and pelleted by centrifugation at 4 °C for 10 min at 300 * g. Cells were lysed in 100 pL of digitonin extraction buffer (10mM 1,4- Piperazinediethanesulfonic acid (PIPES) (pH 6.8), 300mM sucrose, 100mM NaCI, 3mM MgCI2, 5mM EDTA, 0.015% digitonin, 1 mM PMSF) on ice for 10 min, and the lysate was centrifuged at 4 °C for 10 min at 480 * g. The supernatant was kept as the cytosolic fraction. The digitonin-insoluble pellet was then washed once in the same volume of digitonin extraction buffer and spun at 480 * g for 10 min. After the supernatant was discarded, the pellet was resuspended in the same volume of Triton-X-100 extraction buffer (10 mM PIPES (pH 6.8), 300mM sucrose, 100mM NaCI, 3mM MgCI2, 5mM EDTA, 0.5% Triton-X-100, 1 mM PMSF), left on ice for 15 min, and was centrifuged at 5000 * g for 10 min. at 4 °C. The supernatant (membrane/organelle fraction) was discarded, and the Triton-insoluble pellet was lysed in 100 pL of 1x sample buffer to obtain the nuclear fraction. The same cell equivalents of cytosolic and nuclear fractions were resolved on a 10% SDS-PAGE gel, and proteins were analyzed by western blotting.
[00186] Statistics. Statistical analysis was performed using GraphPad™ Prism software (version 6.01 ; GraphPad Software Inc., La Jolla, GA, USA). Results are expressed as means ± SD. The mean comparison was carried out using twotailed Student’s t-test or ANOVA. The number of biologically independent replicates performed for each experiment is indicated in the figure legends.
EXAMPLE 2 - Isolation and characterization of SARS-CoV-2 DVGs
[00187] It order to isolate SARS-CoV-2 DVGs, stock SARS-CoV-2 virus was serially passaged at a high MOI of 3 PFU/cell in Vero E6 cells for 30 passages (Fig. 1A). Assessment of viral titers during different stages of passaging revealed a continuous drop in titers; with the P30 stock showing a 21-fold reduction compared to the P1 stock (Figs. 1 Band 1C).
[00188] Direct RNA sequencing (DRS) of total RNA isolated from P1-, P14-, and P30- infected cells revealed that by P30, a significant proportion of DVGs that arose during serial passaging retained genomic segments spanning Nsp10-Nsp12 (-13.3-16.8 kb) (Fig. 1 D). Northern blot analysis of RNA isolated from infected Vero E6 cells was informative on several points. Firstly, tiling of the CoV-2 genome with a series of radioactive probes revealed the emergence of prominent DVGs of -8 and 7 kb by passage 14 that had retained 5’ end sequences (detected by probe A), the region spanning Nsp12 (the RNA-dependent RNA polymerase (RdRp)) (probe B), and 3’ end sequences (detected by probe G) (Figs.1 E-1 G). Secondly, continued passaging to P30 lead to the loss of the 8 kb DVG and the emergence of prominent 5 kb DVGs (Figs. 1 F-1G, unlabeled arrow). The 5 kb DVGs were first detectable at P20 and persisted up to P30, indicating these were capable of replicating and being packaged in the presence of parental virus. Thirdly, a large population of heterogeneous DVGs was detected by the 3’ end G probe in the latter passages (ie, P20-P30) as smears in the lanes that were absent in the RNA samples from mock-infected cells or that were less abundant in early passage virus stocks (P2-P16) (Figs. 1 F-1G). Nanopore sequencing of RNA from P1 , P15 and P29 infected cells revealed a complex set of DVGs emerging at P15 and P29 (Fig. 1 H). The architecture of the most abundant DVGs from P15 (GI.285 and GI.249) and P29 (GI.616 and GI.50) showed that these: (i) had retained 5’ end sequences, (ii) shared an identical Nsp1-10 junction fusion (see below), (iii) had retained the frameshift site and the Nsp12 ORF, and (iv) had maintained 3’ end sequences (Figs. 11-1 J). [00189] Northern blot analysis of RNA isolated from infected cells at various passages identified prominent -5 kb DVGs from P20 to P30 that had retained Nsp12 and the ORF10/3'UTR regions (Figs. 1 K-1 L). Transcriptmodels constructed from the DRS information and retaining 5' and 3' end sequences (as these regions harbor essential replication signals), indicated loss of a large proportion (>27 kb) of the SARS-CoV-2 genome in P1-infected cells (Figs. 1 M-1O). By P14, a -4.7 kb DVG (GI.535) retaining nucleotides 13,311/13,312-16,841 had emerged. By P30, GI.535 and two related genomes, GI.1634 and GI.1650 (differing only in the 5' end starting location), predominated the DVG population (Fig. 10). To validate the presence of GI.535, a primer pair targeting the 5' and 3' ends (A1 and A2) (Fig. 1 P and Table 4) were used in long-range (LR)-PCRs to amplify the DVGs (Fig. 1Q) and these were cloned and sequenced. GI.535 harbors: (i) an Nsp1-10 in-frame fusion, (ii) sequences spanning Nsp11 , the frameshift signal, and Nsp12, and (iii) an out-of-frame fusion between Nsp13 and the last 116 nts of the N ORF (Fig. 1 P). The appearance of the upstream and downstream junctions (USJ and DSJ) was tracked and it was identified in GI.535 that these appeared to coemerge during serial passaging (Figs. 1 R-1S). GI.535 was the dominant DVG (-83%) throughout P20-P30, indicating stable long-term propagation of this genome (Figs. 1T-1V).
[00190] Nanopore sequencing enabled facile identification and abundance determination of DVG transcript models harboring multiple deletions during SARS-CoV-2 passaging. However, the high error rate (-10%) associated with this technology made it imperative that the sequences of the more abundant DVGs of interest be verified by Sanger sequencing. This was achieved by undertaking long range PGR to amplify the abundant DVGs identified at P14 (Figs. 2A-2D) and P30 (Figs. 2E-2H) using primers anchored to the 5’ and 3’ ends of the SARS-CoV-2 genome (Primers A1/A2). Next, primer pairs to each junction region defined by nanopore sequencing were used to amplify each specific predicted DVG. These were in turn Sanger-sequenced by primer walking. The results indicated that GI.285 and GI.249 were the two most abundant DVGs at P14, and GI.616 and GI.50 were the most abundant DVGs at P30. Surprisingly, it was determined that GI.285, GI.249, GI.616 and GI.50 all share an identical Nsp1-Nsp10 in-frame junction fragment, which resulted from either a 750/13312 or 751/13313 junction fusion (Fig. 3A). Further, it was noted that these four DVGs lost substantial ORFIab coding potential as well as all sgRNA coding regions (from S to ORF8) - with the 3’ junction breakpoint occurring within the N gene. GI.249 harbors an additional deletion within the N ORF. Both GI.616 and GI.50 harbor a missense mutation (344CTC346 [Nsp1 - Leu27] to 344TTC346 [Nsp1 - Phe27]) that is not present in the parental genome, nor in GI.285 or GI.249 (Fig. 3A). GI.616 is distinguished from GI.50 in that it harbors a unique 19 amino acid in-frame deletion within Nsp12 (Fig. 3A). [00191] In order to track the emergence of each of the four DVGs, RT-qPCR was used to analyze the upstream and downstream junctions (USJ and DSJ, respectively) of the viral RNA isolated from infected cells at different passages. The USJ fragment that was common to GI.285, GI.616 and GI.50, and the DSJ fragment that was characteristic of GI.285 were both highly detected at P14-P16 (Figs. 1 F, and 11). However, the DSJ fragment that was characteristic of GI.616 and GI.50 (DSJ-3/DSJ-4) emerged later than the USJ (Figs. 3B-3E). Fragments comprising both the USJ and the DSJ of Gl.50/616 became most prominent in the DVG population from P20 to P30 (Figs. 3F-3G).
[00192] To determine whether the features of Gl.50/616 that were uncovered in the above-mentioned serial passaging experiment (hereinafter referred to as Exp#1 ) could be independently re-acquired, a second serial passaging experiment was conducted under the same conditions (ie. 30 passages in Vero E6 cells at an MOI=3 PFU/cell). In this second independent experiment (hereinafter referred to as Exp#2), it was determined that viral titers decreased in Vero E6 cells by 55-fold when comparing P1 to P30 (Fig. 3H). Northern blot analysis again revealed the emergence of ~5 kb prominent DVGs by P20 which, once again, persisted to P30 (Figs. 3I-3J). Nanopore sequencing showed a DVG architecture similar to that defined in Exp#1 (Figs. 3K, and 3L). Sanger sequencing confirmed the identical Nsp1-10 breakpoint junction as identified in GI.285, GI.249, GI.616 and GI.50, as well as retention of the frameshift site and Nsp12 coding sequences (Fig. 3A). In Exp#2, however, both the USJ and DSJ fragments characteristic of GI.55 appeared to co-emerge during serial passaging, with both junctions becoming most abundant from P20 - P30 (Figs. 1Q-1S). Additionally, it was determined that GI.55 and GI.535 lack the 344TTC346 mutation present in GI.616 and GI.50, which further attests to their independent origin (Fig. 3A).
[00193] To determine whether structural features identified in GI.535 could be independently re-isolated, the serial passaging of SARS-CoV-2 was repeated at high MOI (Exp #2) from the same viral stock used in Exp #1. A 21-fold reduction in viral titers was noted by P30 in Exp #2 (Fig. 3H). Tiling of the SARS-CoV-2 genome using Northern blotting with a series of probes (A-G) revealed the emergence of prominent DVGs of ~6 and 7 kb by passage 14 that had retained 5' proximal sequences (detected by probe A), the region spanning Nsp12 (probe B), and 3' end sequences (detected by probe G) (Figs. 3L-3N). By P20, it was observed that the emergence of ~5 kb DVGs were stably maintained for 10 additional passages (Fig. 30). DRS revealed a pool of DVG structures at P15 that differed from those seen in the first experiment (compare Fig. 3P, P15 to Fig. 1 D, P14). In this experiment, the two most prominent DVGs at P15 were GI.464 (7.2 kb), and GI.384 (5.7 kb) (Figs. 3Q-3S), and these corresponded in size to the genome species that were detected by Northern blotting in P14 (Fig. 3N, probes B and G, indicated by + and *, respectively). The prevalent DVGs from P29 of Exp #2 were found to be similar in structure to those identified in P30 of Exp #1 (compare 30 (P29) to Fig. 1 D (P30)). The top seven most abundant DVGs present in P29 all harbored the identical Nsp1-10 junctions that had been documented in GI.535 (Figs. 3Q-3S). Amplification of the genomes present in P15 and P29 using LR-PCR, followed by Sanger sequencing confirmed the architectures of GI.464, GI.384, GI.616, and GI.50 (Figs. 3P-3S). A 19 amino acid in-frame deletion in Nsp12 is present in GI.616 and distinguishes it from GI.50 (Fig. 3T). LR-PCR revealed the presence of ~7 kb genomes that emerged between P11 and P16 and ~5 kb genomes appearing later between P20 and P30 (Fig. 3U-3V). The abundance of GI.616 and GI.50 was assessed at P20, P25, and P30 and found GI.616 to be the major DVG (25%) present in P25 and P30 (Figs. 3W-3Y). In sum, the structure of the DVGs with the highest relative fitness from both these experiments (i) have retained 5' and 3' end sequences, (ii) harbor the identical Nsp1-10 junction breakpoint, and (iii) maintain Nsp11 , the viral frameshift site, and the Nsp12 coding region (Fig. 3Z).
EXAMPLE 2 - Designation of certain SARS-CoV-2 DVGs as DIPs
[00194] In order to designate a DVG as a DIP, several criteria need to be fulfilled. First, it must be shown that the DVG is capable of interfering with the growth of the parental viral strain. Indeed, for some of the above-mentioned DVGs, this was found to be the case (Figs. 1 B, and 3H). Second, the DVG should be able to undergo packaging in order to be transmitted from cell to cell during virus propagation. This was likely the case for the 5 kb DVGs that emerged at P20 and persisted through serial passaging up to and including P30 (Figs. 1 F-1 G, and 3I-3J). However, in order to formally demonstrate this point, RNA was isolated from Vero E6 cells and supernatant following infection with P1 , P16 and P30 virus stocks from Exp#1 (Fig. 4A). RT-qPCR analysis revealed that only a small amount of cellular GAPDH mRNA was present in the supernatant following infection, which can likely be attributed to cell death (Fig. 4B). In contrast, SARS-CoV-2 genomic (g)RNA was found to be quite abundant in both supernatant and infected cells. Both the USJ and DSJ fragments characteristic of GI.616 and GI.50 were found in RNA isolated from the cytoplasm of P16- and P30-infected cells as well as in the supernatant of the infected cells (Fig. 4B). Northern blots of RNA from infected cells detected the presence of 5 kb DVGs in cells infected with P30 (Fig. 4G, compare lane 3 to 1 ). The presence of DVGs could also be detected in supernatant obtained from P16- and P30-infected cells (Figs. 4C-4D, compare lanes 5 and 6 to 3). These results formally demonstrate that some DVGs were capable of being packaged and released from cells into the supernatant. [00195] The third characteristic that a DVG must display in order to be designated a DIP is a dependence on the parental virus for propagation. To this end, Vero E6 cells were infected at either a MOI of 1 or a MOI of 0.0002 with P2 or P29 viral stocks, and the resulting supernatant was used in 2 additional serial infections. It was expected that, at an MOI of 0.0002, DVGs from P29 would infect cells that have not also been infected by the parental SARS-CoV-2 virus and, therefore, would be lost from the population over time (Figs. 5A-5B). RNA was prepared from the supernatant and cytoplasm of infected cells of the last serial passage (SP3) and analyzed by RT-qPCR. As expected, the control RNA, GAPDH, was detected in cellular RNA preps, but absent from the supernatant samples (Fig. 5C). Also as expected, SARS-CoV-2 gRNA was present in both cytoplasmic and supernatant RNA preps of each of the infected samples, but it was less abundant in the infected P29 samples compared to the P2 samples. While the USJ and DSJ junction fragments common to GI.616 and GI.50 were readily detectable in supernatant and cells that had been infected with at an MOI of 1 the P29 stock, they were absent in the supernatant and cells infected with an MOI of 0.0002 of the same stock (Fig. 5C). These results indicate that the DVGs present in P29 could not replicate on their own. Therefore, at this juncture, these DVGs were tentatively classified these as DIP. DVGs were recovered only from cells and media infected with the P29 (MOI = 1 ) stock (Fig. 5D). No DVGs were detected in extracts or media from cells infected at an MOI = 0.0002. Thus, the prominent DVGs are only propagated in the presence of helper virus, and their detection following serial passaging also indicates that they are packaged. Henceforth, these DVGs are referred to as SARS-CoV-2 DI particles.
EXAMPLE 3 - Construction of recombinant, infectious DI SARS-CoV-2 genomes
[00196] To determine whether specific synthetic, recombinant DVG genomes could function as DIPs, sequences corresponding to GI.50, GI.55 and GI.616 were cloned downstream of the T7 RNA polymerase promoter and appended a 3’ poly(A) tail (Fig. 6A). Eight hours following infection of Vero cells with SARS-CoV-2, in vitro transcribed RNA corresponding to Renilla luciferase (RLuc; negative control), GI.50, GI.55, or GI.616 were transfected into 293T/Ace cells. After 22 hours, the supernatant was obtained and serially passaged four times (P1 - P4). RNA was isolated from the supernatant of cells prior to serial passaging (P0) and from the supernatant of the P2 and P4 infected cells (Fig. 6A). RT-qPCR analysis showed that cellular GAPDH, actin, and 18S rRNA levels were relatively constant across all the samples (Fig. 6B). As expected, RLuc mRNA was detectable in P0 cells, but not in P2 or P4 cells, and SARS-CoV-2 gRNA was readily detected in all infected cells. The USJ and DSJ, which are unique characteristics of the DVG genomes, were both present in P0 cells that had been transfected with the synthetic, recombinant DVG genomes, as well as in the P2 and P4 cells (Fig. 6B). In the absence of any SARS-CoV-2 virus infection, no GI.50, GI.55 or GI.616 genomes were detectable in the P2 samples, which illustrates the dependence of these DVGs on the parental virus for propagation (Fig. 60). All three full- length synthetic DVG genomes were recovered from P4 infected cells (Fig 6D) and were associated with significantly reduced SARS-CoV-2 titers (Fig. 6E). Taken together, these results demonstrate that recombinant RNA derived from SARS-CoV-2 DVGs is capable of replicating and being packaged in the presence of parental SARS-CoV-2. Further, this recombinant RNA is capable of attenuating the replication of the parental SARS-CoV-2.
[00197] In order to assess whether synthetic DI genomes derived from SARS-CoV-2 could be used as gene delivery vectors, an EMCV-driven Renilla luciferase (EMCV/Ren) cassette was genetically engineered at the DSJ in GI.55 and GI.616, between Nsp13 and the N ORF (Fig. 6F). Following SARS-CoV-2 infection and DI RNA transfection into Vero cells, the supernatant hat was obtained was serially passaged five times. In mock-infected cells, all transfected RNA constructs produced detectable levels of luciferase activity (Fig. 6G). In transfected Vero E6 cells that received the virus (PO), luciferase activity was also readily detectable, although the synthetic DI RNA/RLuc constructs produced less protein (Fig. 6G). Lysates prepared from P3 and P4 cells showed no activity from serially passaged supernatant from mock- or RLuc mRNA-transfected cells (Fig 6C) In contrast, supernatant from cells having initially received GI.50/RLuc or GI.616/RLuc yielded strong lucifersae activity (Fig. 6G). This was associated with a 13-20 fold reduction in viral titers at P3 and P4 (Fig. 6H). These results indicate that the DI genomes identified herein can be used not only to blunt a SARS-CoV-2 infection but can also be used as a gene delivery vector.
EXAMPLE 4 - Synthetic, recombinant DI genomes exhibit long-term stability and attenuate SARS-CoV-2 replication.
[00198] To formally demonstrate that the DI genomes that were isolated were responsible for attenuating SARS-CoV-2 replication, the sequences corresponding to GI.50 and GI.616 were placed under control of the T7 RNA polymerase promoter and appended a 3' poly(A) tail (Fig. 7A). Synthetic DI RNAs and RLuc mRNA (negative control) were generated in vitro. Eight hours following infection of Vero E6 cells with SARS-CoV-2, RLuc, GI.50, and GI.616 RNA were transfected into cells. Twenty-two hours later, the virus was collected and serially passaged four times (Fig. 7A). Plaque assays showed that viral titers were reduced by 10- 20-fold in cells that received recombinant DI RNA following infection, whereas no reduction was apparent in cells having received RLuc mRNA (Fig. 7B). Full-length synthetic DI genomes were recovered by LRPCR from RNA of SP4-infected cells (Fig. 70). Probing cell lysates with a-Nsp1 antibodies revealed the presence of Nsp1 in virus infected cells (Fig. 7D, compare lanes 2-4 to lane 1 ). Cells that received the virus from GI.616 transfected cells also expressed an immune-reactive protein whose molecular mass is consistent with it being an Nsp1-10 fusion product (Fig. 70, compare lane 4 to 3). RNA from P0, SP2, and SP4 infections was analyzed by RT-qPCR for the presence of SARS-CoV-2 gRNA and DI genomes (Fig. 7E [raw Ct values] and Fig. 7F [data normalized to GAPDH mRNA levels and expressed relative to CoV-2 gRNA levels]). As expected, RLuc mRNA was present in P0 transfected cells, but not in SP2- or SP4-infected cells (Fig. 7E and Fig. 7F). SARS-CoV-2 gRNA was readily detected in all infected cells. The USJ and DSJ, unique characteristics of the DI genomes, were present in transfected (P0) cells, as well as in SP2- and SP4-infected cells (Fig. 7E). GAPDH mRNA and 18 S rRNA levels were similar across samples (Fig. 7G). In the absence of SARS-CoV-2 virus, GI.50 and GI.616 genomes were not present in SP2 samples, consistent with DI replication being dependent on the parental virus (Fig. 7H). These results indicate that synthetic, recombinant DI RNA can conditionally and stably propagate in the presence of parental SARS-CoV-2 where they attenuate viral replication in a post-infection setting.
[00199] To assess if Dis could be used as conditional gene delivery vehicles, an EMCV- driven Renilla luciferase (EMCV/RLuc) or transcription regulatory sequence (TRS/RLuc) cassette reporter was inserted into the DSJ of GI.616 (Fig. 7I). Vero E6 cells were infected with SARS-CoV-2, and DI RNA transfections performed 1 hpi. This was then followed by four serial passages. In cells receiving SP4 virus from the RLuc transfections, only background levels of luciferase activity were detected (Fig. 7J). In contrast, cells infected with SP4 virus from GI.616-EMCV/RLuc transfections produced significant luciferase activity (Fig. 7J). However, the highest levels of luciferase were from GI.616-TRS/RLuc samples which were 320-fold higher than cells containing GI.616-EMCV/RLuc Dis (Fig. 7J). The presence of recombinant GI.616-EMCV/RLuc or GI.616-TRS/RLuc genomes reduced SARS-CoV-2 titers 15- and 30-fold, respectively (Fig. 7K). We confirmed that GI.616-TRS/RLuc produced a subgenomic mRNA containing the viral 5' TRS-L end sequences by RT-PCR using primers targeting TRS-L and the renilla ORF (Fig. 7L, lane 3). Taken together, these results indicate that synthetic versions of the DI genomes identified herein can be used as conditional gene delivery vectors to inhibit SARS-CoV-2 replication.
EXAMPLE 5 - Recombinant DI genomes interfere with SARS-CoV-2 replication
[00200] It was next sought to query the mechanism by which GI.616 restricts viral replication. Following infection and transfection of Vero E6 cells, virus was serially passaged three times, and levels of viral RNA in the media and cells were determined at SP2 and SP3 (Fig. 8A). In P2-infected cells, GI.616 reduced SARS-CoV-2 gRNA levels compared to RLuc controls (Figs. 8B-8C). Levels of SARS-CoV-2 gRNA in P2-infected cells relative to virions present in SP3 media were then compared. The presence of GI.616 did not affect the packaging or release of SARS-CoV-2 gRNA from cells (Fig. 8D). In addition, GI.616 was packaged and released at the same efficiency as SARS-CoV-2 gRNA (Fig. 8D). The presence of GI.616 did not affect the transmission of SARS-CoV-2 gRNA (Fig. 8E). However, GI.616 genomes transmitted at a rate four-fold higher compared to SARS-CoV-2 gRNA (Fig. 8E). Taken together, these results indicate that robust replication of GI.616 during the early stages of infection (by 4 h) is associated with reduced SARS-CoV-2 gRNA levels over the course of infection.
EXAMPLE 6 - SARS-CoV-2 Dis encode an Nsp1-10 fusion that inhibits viral replication
[00201] SARS-CoV-2 Nsp1 is a multifunctional protein that has been implicated in blocking host translation, degradation of cellular mRNAs, and inhibition of nucleo-cytoplasmic mRNA export. To determine if Nsp1-10 could be detected in infected cells (Fig. 9A), Western blots were performed on extracts from Vero E6 cells infected with P2, P15, or P30 stocks. Results from these experiments showed that Nsp1 (—20 kDa) was present in infected cells (Fig. 9B, bottom arrow), whereas a larger ~30 kDa protein that cross-reacted with antibodies to Nsp1 was present in P15- and P30-infected cells (top arrow). This protein could also be detected using an antibody targeting the C-terminal domain of Nsp10, which revealed an immunoreactive protein at ~30 kDa (Fig. 9C, top arrow). The Nsp1-10 fusion protein encoded by the prominent Dis was confined predominantly to the cytoplasm when overexpressed in uninfected cells (Fig. 9D).
[00202] The Nsp1 C-terminal domain is essential for blocking translation as it interacts with the mRNA entry channel to inhibit cellular protein synthesis. However, this domain is absent from the Nsp1-10 fusion protein (Fig. 9E). Consequently, Nsp1 , but not Nsp1-10, inhibited cellular translation as assessed by polysome profiling (Figs. 9F-9G). Nsp1-10, unlike Nsp1 , did not co-migrate with 40 S ribosomes in polysome gradients (Figs. 9F-9G). These data are consistent with what was observed for a previously described Nsp1(KH/AA) mutant that does not block translation (Figs. 9H-9I). Nsp1 , but neither Nsp1-10 nor Nsp1(KH/AA), inhibited 35S-Met/Cys incorporation into the nascent polypeptide chain (Figs. 9J-9K). Nsp1-10 was unable to rescue Nsp1-mediated inhibition of translation in cells (Fig. 9J) or in vitro (Figs. 9L-9M).
[00203] To assess the requirements of the Nsp1-10 region for DI replication, two deletion mutants were generated, ANTD-Nsp1 and A2NTD-Nsp1 , in GI.616 (Fig. 9C). Propagation of the mutant Dis was compromised after co-passaging with the parental virus for two passages (Fig. 9N [raw Ct values] and Fig. 90 [data normalized to GAPDH mRNA levels and expressed relative to CoV-2 gRNA levels]) and parental viral titers were not significantly affected (Fig. 9P). Ectopic expression of Nsp1-10 in 293 T/ACE2 cells reduced SARS-CoV-2 titers by 25-fold (Figs. 9Q-9R). In vitro, recombinant Nsp1 was shown to be capable of inhibiting cap-dependent translation (Figs. 10A-10F). However, the KH/AA Nsp1 mutant was inactive. Likewise, Nsp1-10 did not display any host translation inhibition activity. The effect of Nsp1-10 was selective in that it did not reduce titers of Dengue type 2 virus (Fig. 10G). The data supports the conclusion that Nsp1-10 is a potent inhibitor of SARS-CoV-2 replication. When Nsp1 and Nsp1-10 were co-transfected into cells, there was no effect on Nsp1 mediated inhibition of translation (Fig. 10H). Lastly, infection of 293/ AC E2 cells stably expressing Nsp1-10 with SARS-CoV2 clearly diminished viral yields 25-fold, relative to control cells (Fig. 101).
EXAMPLE 7 - Genetic Interference by DIPs in vivo
[00204] In order to demonstrate genetic interference of SARS-CoV-2 by DI particles in vivo, hACE2 mice were intranasally infected with 5 x 104 PFU of stock virus, 5 x 104 PFU of P1 virus, 5 x 103 PFU of P30-virus, or with a combination of 5 x 104 PFU of P1- virus and 5 x 103 PFU P30-virus (Fig. 11 A). All animals were monitored daily for clinical symptoms and body weight changes and were sacrificed when body weights dropped <20% during the experiment or on day 10. Mice inoculated with stock or P1 virus had to be sacrificed by day 6 post-infection whereas mice receiving only P30 were sacrificed on day 7 (Fig. 11 B). Mice inoculated with stock, P1 , or P30 virus attained a grade 4 clinical score (Fig. 11C). Of the mice receiving the combination of P1 + P30, three were sacrificed on day due to 20% body weight lost but two survived beyond this time point and by day 10 were back to their original weight (Fig. 11 D). None of the five mice receiving P1 + P30 showed a clinical score above 2 (Figs. 11B-11C). These experiments demonstrate the protective effect of P30-virus on SARS-CoV-2 infection of susceptible hACE2 mice in vivo.
EXAMPLE 7 - GI.616 encoded Nsp12 (A19aa) is inactive for polymerase activity.
[00205] The impact of the Nsp12 (A19aa) mutation in GI.616 might have on Nsp12 activity was evaluated. Based on the structure of Nsp12, the deletion of 19 amino acids was expected to shorten the distance between the finger region and palm domain of the protein and alter RNA binding (Fig. 12A). Using a polymerase extension assay where activity is assessed using a 4-mer primer, we monitored the appearance of a 14-nts product (Fig. 12B, lane 1 ). The previously described Nsp12 (SNN) active site mutant was inactive in this assay (compare lanes 13-18 to 1 )36. Similarly, the Nsp12(A19aa) mutant was also compromised for polymerase activity (compare lanes 19-24 to 1 ). The presence of Nsp12 (SNN) (lanes 2- 6) or Nsp12 (A19aa) (lanes 8-12) in reactions containing WT RdRP did not compromise RdRP activity, attesting to a lack of dominant-negative behavior.
DISCUSSION
[00206] It was found that a salient feature present in coronavirus Dis is the retention of sequences at the 5' and 3' ends of the genome - a finding consistent with stemloops at these locations being essential for replication. A common, conserved upstream junction element, fusing Nsp1 to Nsp10 was present in 80-90% percent of late passage DVGs. Nsp1-10 present at P0 or arising during propagation, was clearly positively selected for during serial passaging in two independent instances.
[00207] Although the Nsp1-10 fusion arose in interferon-deficient Vero cells, its function is not restricted to this context since the expression of Nsp1-10 in ACE2-expressing 293 T cells attenuated viral replication (Fig. 9R). The Nsp1-10 protein thus appears to represent a Dl-encoded protein that attenuates helper virus replication. Unlike Nsp1 , Nsp1-10 did not interfere with cellular translation (Figs. 9F, 9G, and 9J). Nsp1 is a multifunctional protein that has also been implicated in host mRNA cleavage, and blockade of mRNA export. Of note, in the present Examples there was no prominent detection of stable defective viral genomes that were <5 kbp in length by Northern blotting (Fig. 1 K, 1 L and 3M-3O) nor as prevalent transcript models in the nanopore sequencing data from late passages of both experiments (Fig. 1 M-1 R). These results indicate that the ~5 kb genomes that were characterized are stable to long-term propagation in the presence of the parental virus.
[00208] Defective interfering particles can cause cyclical changes in viral titers since they not only compete with but also rely on, parental viruses for propagation. In the presence of DI, parental virus levels will drop and reach a local minimum, DI levels subsequently do the same as they are dependent on the parental virus for their replication. This, in turn, leads to parental virus levels peaking as there are minimal DI particles available for competition. With increasing passage number, this results in cyclic changes in parental virus levels and DI levels where the peak in DI levels is superimposed with a trough in parental virus levels and vice versa. Indeed the DI genome replication and encapsidation are two critical parameters that affect wild type virus outcome. Therefore, one successful therapeutic strategy is to deliver the RNA encoding the DI particle intranasally to treat coronavirus infections.
REFERENCES Lai MM. RNA recombination in animal and plant viruses. Microbiol Rev. 1992;56(1):61-79. Liao CL, Lai MM. RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments. J Virol. 1992;66(10):6117-24. Furuya T, Macnaughton TB, La Monica N, Lai MM. Natural evolution of coronavirus defective-interfering RNA involves RNA recombination. Virology. 1993;194(1 ):408-13. Viehweger A, Krautwurst S, Lamkiewicz K, Madhugiri R, Ziebuhr J, Holzer M, et al. Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis. Genome Res. 2019;29(9): 1545-54. Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The Architecture of SARS-CoV- 2 Transcriptome. Cell. 2020;181(4):914-21 e10. Gribble J, Stevens LJ, Agostini ML, Anderson-Daniels J, Chappell JD, Lu X, et al. The coronavirus proofreading exoribonuclease mediates extensive viral recombination. PLoS Pathog. 2021;17(1):e1009226. Noble S, Dimmock NJ. Characterization of putative defective interfering (DI) A/WSN RNAs isolated from the lungs of mice protected from an otherwise lethal respiratory infection with influenza virus A/WSN (H1 N1 ): a subset of the inoculum DI RNAs. Virology. 1995;210(1 ):9-19. Dimmock NJ, Rainsford EW, Scott PD, Marriott AC. Influenza virus protecting RNA: an effective prophylactic and therapeutic antiviral. J Virol. 2008;82(17):8570-8. Yang Y, Lyu T, Zhou R, He X, Ye K, Xie Q, et al. The Antiviral and Antitumor Effects of Defective Interfering Particles/Genomes and Their Mechanisms. Front Microbiol. 2019; 10: 1852. Rezelj VV, Carrau L, Merwaiss F, Levi LI, Erazo D, Tran QD, et al. Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts. Nat Commun. 2021 ;12(1 ):2290. Levi LI, Rezelj VV, Henrion-Lacritick A, Erazo D, Boussier J, Vallet T, et al. Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes. PLoS Pathog. 2021 ;17(2):e1009110. van der Most RG, Bredenbeek PJ, Spaan WJ. A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs. J Virol. 1991 ;65(6):3219-26. Kim YN, Lai MM, Makino S. Generation and selection of coronavirus defective interfering RNA with large open reading frame by RNA recombination and possible editing. Virology. 1993; 194(1 ):244-53. Lin YJ, Lai MM. Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication. J Virol. 1993;67(10):6110-8. Lin YJ, Zhang X, Wu RC, Lai MM. The 3' untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. J Virol. 1996;70(10):7236-40. Makino S, Shieh OK, Keck JG, Lai MM. Defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral RNAs. Virology.
1988; 163(1): 104-11. Goebel SJ, Miller TB, Bennett GJ, Bernard KA, Masters PS. A hypervariable region within the 3' cis-acting element of the murine coronavirus genome is nonessential for RNA synthesis but affects pathogenesis. J Virol. 2007;81(3):1274-87. Kim YN, Makino S. Characterization of a murine coronavirus defective interfering RNA internal cis-acting replication signal. J Virol. 1995;69(8):4963-71. Makino S, Fujioka N, Fujiwara K. Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus. J Virol. 1985;54(2):329-36. Makino S, Yokomori K, Lai MM. Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal. J Virol. 1990;64(12):6045-53. Mendez A, Smerdou C, Izeta A, Gebauer F, Enjuanes L. Molecular characterization of transmissible gastroenteritis coronavirus defective interfering genomes: packaging and heterogeneity. Virology. 1996;217(2):495-507. Izeta A, Smerdou C, Alonso S, Penzes Z, Mendez A, Plana-Duran J, et al.
Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes. J Virol. 1999;73(2): 1535-45. Chang RY, Hofmann MA, Sethna PB, Brian DA. A cis-acting function for the coronavirus leader in defective interfering RNA replication. J Virol. 1994;68(12):8223- 31. Chang RY, Brian DA. cis Requirement for N-specific protein sequence in bovine coronavirus defective interfering RNA replication. J Virol. 1996;70(4):2201-7. Williams GD, Chang RY, Brian DA. A phylogenetically conserved hairpin-type 3' untranslated region pseudoknot functions in coronavirus RNA replication. J Virol. 1999;73(10):8349-55. Raman S, Bouma P, Williams GD, Brian DA. Stem-loop III in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication. J Virol. 2003;77(12):6720-30. Brown CG, Nixon KS, Senanayake SD, Brian DA. An RNA stem-loop within the bovine coronavirus nsp1 coding region is a cis-acting element in defective interfering RNA replication. J Virol. 2007;81(14):7716-24. Thiel V, Siddell SG, Herold J. Replication and transcription of HCV 229E replicons. p6. Adv Exp Med Biol. 1998;440:109-13. Yao S, Narayanan A, Majowicz SA, Jose J, Archetti M. A synthetic defective interfering SARS-CoV-2. PeerJ. 2021 ;9:e11686. Schubert K, Karousis ED, Jomaa A, Scaiola A, Echeverria B, Gurzeler LA, et al. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nat Struct Mol Biol. 2020;27(10):959-66. Sambrook J, Russell DW. Molecular cloning : a laboratory manual. 3rd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2001. Novac O, Guenier AS, Pelletier J. Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res. 2004;32(3):902-15.

Claims

- 65 - WHAT IS CLAIMED IS:
1. An isolated polypeptide capable of limiting the replication of a coronavirus, the isolated polypeptide comprising a fusion of at least two polypeptides encoded by viral open reading frames (ORFs) of ORF1a of a coronavirus.
2. The isolated polypeptide of claim 1 , wherein the at least two polypeptides comprise Nsp1 and Nsp10 and the fusion comprises a Nsp1 moiety and a Nsp10 moiety.
3. The isolated polypeptide of claim 2, wherein the Nsp1 moiety comprises the amino acid sequence of SEQ ID NO: 3, a variant of the amino acid sequence of SEQ ID NO: 1 or 3 and/or a fragment of the amino acid sequence of SEQ ID NO.: 1 or 3.
4. The isolated polypeptide of claim 2 or 3, wherein the Nsp10 moiety comprises the amino acid sequence of SEQ ID NO: 4, a variant of the amino acid sequence of SEQ ID NO: 2 or 4 and/or a fragment of the amino acid sequence of SEQ ID NO: 2 or 4.
5. The isolated polypeptide of any one of claims 1 to 4, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 5, a variant of the amino acid sequence of SEQ ID NO: 5 and/or a fragment of SEQ ID NO: 5.
6. An isolated nucleic acid molecule encoding the polypeptide defined in any one of claims 1 to 5.
7. The isolated nucleic acid molecule of claim 6 comprising one or more ribonucleic acid residues.
8. The isolated nucleic acid molecule of claim 7 being a messenger RNA molecule.
9. The isolated nucleic acid molecule of claim 6 comprising one or more deoxyribonucleic acid residues.
10. The isolated nucleic acid molecule of claim 9 being a DNA molecule.
11. The isolated nucleic acid molecule of any one of claims 6 to 10 further comprising a heterologous gene.
12. The isolated nucleic acid molecule of claims 11 , wherein the heterologous gene is expressed using an internal ribosomal entry site (IRES).
13. The isolated nucleic acid molecule of claims 11 , wherein the heterologous gene is expressed using a coronavirus transcription regulatory sequence (TRS).
14. The isolated nucleic acid molecule of claim 11 , wherein the heterologous gene encodes a killswitch, an interferon, a short hairpin RNA (shRNA), a single chain antibody, and/or Cas9.
15. An isolated nucleic acid molecule comprising a defective viral genome (DVG) encoding a therapeutic interfering particle (TIP), wherein the therapeutic interfering particle:
(i) is capable of limiting the replication of a human coronavirus; - 66 -
(ii) is replication defective and can be replicated in the presence of a helper virus;
(iii) is defective for packaging and can be packaged in the presence of the helper virus;
(iv) is capable of being enriched upon a plurality of passage in a cell infected with the coronavirus and/or the helper virus at a multiplicity of infection equal to or greater than 1 ; and
(v) encodes the polypeptide defined in any one of claims 1 to 5 and/or comprises the nucleic acid molecule defined in any one of claims 6 to 14. The isolated nucleic acid molecule of claim 15, wherein the therapeutic interfering particle is a defective interfering particle (DIP). The isolated nucleic acid molecule of claim 15 or 16, wherein the coronavirus is the helper virus. The isolated nucleic acid molecule of any one of claims 15 to 17, wherein the coronavirus and/or the helper virus is from the alpha genus. The isolated nucleic acid molecule of claim 18, wherein the coronavirus and/or the helper virus is 229E or NL63. The isolated nucleic acid molecule of claim 15 to 17, wherein the coronavirus and/or the helper virus is from the beta genus. The isolated nucleic acid molecule of claim 20, wherein the human coronavirus and/or the helper virus is OC43, HKU1 , SARS-CoV, MERS-CoV, or SARS-CoV2. The isolated nucleic acid molecule of claim 15 to 17, wherein the coronavirus and/or the helper virus is from the gamma genus. The isolated nucleic acid molecule of claim 15 to 17, wherein the coronavirus and/or the helper virus is from the delta genus. The isolated nucleic acid molecule of any one of claims 13 to 23 comprising, when compared to the nucleic acid sequence of the genome of the coronavirus or of the helper virus, a first deletion in an open reading frame 1 a. The isolated nucleic acid molecule of claim 24, wherein the first deletion starts at a first start position corresponding to position 749 of GenBank accession number NC_045512. The isolated nucleic acid molecule of claim 24 or 25, wherein the first deletion ends at a first end position corresponding to position 13311 of GenBank accession number NC_045512. The isolated nucleic acid molecule of any one of claims 24 to 26, wherein the first deletion encompasses the nucleic acid sequence encoding at least one of Nsp2, Nsp3, Nsp4, Nsp5, Nsp6, Nsp7, Nsp8, or Nsp9. The isolated nucleic molecule of any one of claims 15 to 27, wherein the TIP is capable of limiting the titer of the coronavirus and/or of the helper virus by at least one log or more. - 67 - The isolated nucleic acid molecule of any one of claims 13 to 27 having the nucleic acid sequence of GI.285, GI.249, GI.616, GI.50, GI.55 or GI.535. A therapeutic interfering particle comprising the polypeptide defined in any one of claims 1 to 5 and/or the nucleic acid molecule defined in any one of claims 6 to 29. A pharmaceutical composition comprising (i) the polypeptide defined in any one of claims 1 to 5 and/or the nucleic acid molecule defined in any one of claims 6 to 29 and (ii) a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 31 being formulated for nasal administration. A method of treating a coronavirus infection in a subject in need thereof, the method comprises administering a therapeutically effective amount of the polypeptide defined in any one of claims 1 to 5, the nucleic acid molecule defined in any one of claims 6 to 26 and/or the pharmaceutical composition of claim 31 or 32 to the subject so as to reduce the replication of the coronavirus. The method of claim 33, wherein the subject is a human or animal subject.
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CHATURVEDI SONALI; VASEN GUSTAVO; PABLO MICHAEL; CHEN XINYUE; BEUTLER NATHAN; KUMAR ARJUN; TANNER ELIZABETH; ILLOUZ SYLVIA; RAHGOS: "Identification of a therapeutic interfering particle—A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance", CELL, vol. 184, no. 25, 10 November 2021 (2021-11-10), Amsterdam NL , pages 6022, XP086893886, ISSN: 0092-8674, DOI: 10.1016/j.cell.2021.11.004 *

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