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Journal of General Virology header logo Journal of General Virology

Volume 106, Issue 1

A yeast-assembled, plasmid-launched reverse genetics system for the murine coronavirus MHV-A59 No Access

Abstract

The murine hepatitis virus (MHV) is an important model system for studying coronavirus (CoV) molecular and cell biology. Despite this, few reagents for MHV are available through repositories such as ATCC or Addgene, potentially limiting the widespread adoption of MHV as a tractable model system. To overcome some challenges inherent in the existing MHV reverse genetics systems, we developed a plasmid-launched transformation-associated recombination (TAR) cloning-based system to assemble the MHV (strain A59; MHV-A59) genome. Following assembly in yeast, virus replication was launched by transfecting the fully assembled genome into HEK-293T cells. MHV-A59 recovered using this TAR cloning-based approach (WT MHV-A59) replicated with kinetics identical to the virus recovered using a ligation- and T7-based approach (WT MHV-A59). Additionally, WT MHV-A59 can be detected at least 10 h post-transfection without requiring additional nucleocapsid (N) provided in trans. Lastly, we demonstrated the tractability of this TAR cloning-based system by recovering MHV-A59 expressing an 11 amino acid-containing HiBiT tag fused to the C-terminus of spike (S). While this virus, S MHV-A59, replicated with reduced kinetics compared to WT MHV-A59, the kinetics of virion production could be measured over time directly from the supernatant. This report represents the first plasmid-launched, TAR cloning-based system for MHV-A59. Furthermore, it describes a new reporter virus that could be used to study early steps during MHV-A59 entry and be used in the screening of antiviral compounds. To support future research with MHV-A59, we have made the necessary plasmids for this system available through ATCC.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): coronavirus , HiBiT , MHV-A59 , reverse genetics , RNA virus and TAR cloning
Funding
This study was supported by the:
  • Appalachian College Association
    • Principle Award Recipient: EverettClinton Smith
  • Appalachian College Association
    • Principle Award Recipient: CadeE. Sterling
© 2025 The Authors
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References

  1. Sterling CE, Wilson N, Harris D, Smith E. A yeast-assembled, plasmid-launched reverse genetics system for the murine coronavirus MHV-A59 Microbiology Society 2025 [View Article]
     [Google Scholar]
  2. Woo PCY, de Groot RJ, Haagmans B, Lau SKP, Neuman BW et al. ICTV virus taxonomy profile: coronaviridae. J Gen Virol 2023; 104:001843 [View Article]
     [Google Scholar]
  3. Eckerle LD, Lu X, Sperry SM, Choi L, Denison MR. High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. J Virol 2007; 81:12135–12144 [View Article] [PubMed]
     [Google Scholar]
  4. Ogando NS, Zevenhoven-Dobbe JC, van der Meer Y, Bredenbeek PJ, Posthuma CC et al. The enzymatic activity of the nsp14 exoribonuclease is critical for replication of MERS-CoV and SARS-CoV-2. J Virol 2020; 94:01246–20 [View Article] [PubMed]
     [Google Scholar]
  5. Minskaia E, Hertzig T, Gorbalenya AE, Campanacci V, Cambillau C et al. Discovery of an RNA virus 3’->5’ exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc Natl Acad Sci USA 2006; 103:5108–5113 [View Article] [PubMed]
     [Google Scholar]
  6. Schaad MC, Stohlman SA, Egbert J, Lum K, Fu K et al. Genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand RNA synthesis. Virol 1990; 177:634–645 [View Article] [PubMed]
     [Google Scholar]
  7. Koolen MJM, Osterhaus ADME, Van Steenis G, Horzinek MC, van der Zeijst BAM. Temperature-sensitive mutants of mouse hepatitis virus strain A59: isolation, characterization and neuropathogenic properties. Virol 1983; 125:393–402 [View Article]
     [Google Scholar]
  8. Bos EC, Dobbe JC, Luytjes W, Spaan WJ. A subgenomic mRNA transcript of the coronavirus mouse hepatitis virus strain A59 defective interfering (DI) RNA is packaged when it contains the DI packaging signal. J Virol 1997; 71:5684–5687 [View Article] [PubMed]
     [Google Scholar]
  9. Cologna R, Spagnolo JF, Hogue BG. Identification of nucleocapsid binding sites within coronavirus-defective genomes. Virol 2000; 277:235–249 [View Article] [PubMed]
     [Google Scholar]
  10. Kim Y-N, Jeong YS, Makino S. Analysis of cis-acting sequences essential for coronavirus defective interfering RNA replication. Virol 1993; 197:53–63 [View Article] [PubMed]
     [Google Scholar]
  11. 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; 67:6110–6118 [View Article] [PubMed]
     [Google Scholar]
  12. Makino S, Fujioka N, Fujiwara K. Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus. J Virol 1985; 54:329–336 [View Article] [PubMed]
     [Google Scholar]
  13. 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; 65:3219–3226 [View Article] [PubMed]
     [Google Scholar]
  14. Baric RS, Fu K, Schaad MC, Stohlman SA. Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups. Virol 1990; 177:646–656 [View Article] [PubMed]
     [Google Scholar]
  15. Koetzner CA, Parker MM, Ricard CS, Sturman LS, Masters PS. Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination. J Virol 1992; 66:1841–1848 [View Article] [PubMed]
     [Google Scholar]
  16. Kuo L, Godeke G-J, Raamsman MJB, Masters PS, Rottier PJM. Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier. J Virol 2000; 74:1393–1406 [View Article] [PubMed]
     [Google Scholar]
  17. van der Most RG, Heijnen L, Spaan WJ, de Groot RJ. Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs. Nucleic Acids Res 1992; 20:3375–3381 [View Article] [PubMed]
     [Google Scholar]
  18. Masters PS, Koetzner CA, Kerr CA, Heo Y. Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus. J Virol 1994; 68:328–337 [View Article] [PubMed]
     [Google Scholar]
  19. Yount B, Denison MR, Weiss SR, Baric RS. Systematic assembly of a full-length infectious cDNA of mouse hepatitis Virus strain A59. J Virol 2002; 76:11065–11078 [View Article]
     [Google Scholar]
  20. Yount B, Curtis KM, Baric RS. Strategy for systematic assembly of large RNA and DNA genomes: transmissible gastroenteritis virus model. J Virol 2000; 74:10600–10611 [View Article] [PubMed]
     [Google Scholar]
  21. Coley SE, Lavi E, Sawicki SG, Fu L, Schelle B et al. Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo. J Virol 2005; 79:3097–3106 [View Article] [PubMed]
     [Google Scholar]
  22. Casais R, Thiel V, Siddell SG, Cavanagh D, Britton P. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J Virol 2001; 75:12359–12369 [View Article] [PubMed]
     [Google Scholar]
  23. Thiel V, Herold J, Schelle B, Siddell SG. Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J Gen Virol 2001; 82:1273–1281 [View Article] [PubMed]
     [Google Scholar]
  24. Karstentischer B, von Einem J, Kaufer B, Osterrieder N. Two-step Red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. BioTechniques 2006; 40:191–197 [View Article]
     [Google Scholar]
  25. Fehr AR, Athmer J, Channappanavar R, Phillips JM, Meyerholz DK et al. The nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis. J Virol 2015; 89:1523–1536 [View Article] [PubMed]
     [Google Scholar]
  26. Oldfield LM, Grzesik P, Voorhies AA, Alperovich N, MacMath D et al. Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods. Proc Natl Acad Sci USA 2017; 114:E8885–E8894 [View Article] [PubMed]
     [Google Scholar]
  27. Vashee S, Stockwell TB, Alperovich N, Denisova EA, Gibson DG et al. Cloning, assembly, and modification of the primary human Cytomegalovirus isolate toledo by yeast-based transformation-associated recombination. mSphere 2017; 2:e00331-17 [View Article] [PubMed]
     [Google Scholar]
  28. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang R-Y et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 2010; 329:52–56 [View Article] [PubMed]
     [Google Scholar]
  29. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 2008; 319:1215–1220 [View Article] [PubMed]
     [Google Scholar]
  30. Cao H, Gu H, Kang H, Jia H. Development of a rapid reverse genetics system for feline coronavirus based on TAR cloning in yeast. Front Microbiol 2023; 14:1141101 [View Article] [PubMed]
     [Google Scholar]
  31. Noskov VN, Koriabine M, Solomon G, Randolph M, Barrett JC et al. Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning. Nucleic Acids Res 2001; 29:e32 [View Article] [PubMed]
     [Google Scholar]
  32. Thi Nhu Thao T, Labroussaa F, Ebert N, V’kovski P, Stalder H et al. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature 2020; 582:561–565 [View Article] [PubMed]
     [Google Scholar]
  33. Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips APR et al. Designer diatom episomes delivered by bacterial conjugation. Nat Commun 2015; 6:6925 [View Article] [PubMed]
     [Google Scholar]
  34. Smith EC, Case JB, Blanc H, Isakov O, Shomron N et al. Mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity. J Virol 2015; 89:6418–6426 [View Article] [PubMed]
     [Google Scholar]
  35. Noskov V, Kouprina N, Leem S-H, Koriabine M, Barrett JC et al. A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast. Nucleic Acids Res 2002; 30:8e–88 [View Article]
     [Google Scholar]
  36. Blount BA, Driessen MRM, Ellis T. GC Preps: fast and easy extraction of stable yeast genomic DNA. Sci Rep 2016; 6:26863 [View Article] [PubMed]
     [Google Scholar]
  37. Chen W, Baric RS. Molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence. J Virol 1996; 70:3947–3960 [View Article] [PubMed]
     [Google Scholar]
  38. Rihn SJ, Merits A, Bakshi S, Turnbull ML, Wickenhagen A et al. A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research. PLoS Biol 2021; 19:e3001091 [View Article] [PubMed]
     [Google Scholar]
  39. Amarilla AA, Sng JDJ, Parry R, Deerain JM, Potter JR et al. A versatile reverse genetics platform for SARS-CoV-2 and other positive-strand RNA viruses. Nat Commun 2021; 12:3431 [View Article]
     [Google Scholar]
  40. Kouprina N, Annab L, Graves J, Afshari C, Barrett JC et al. Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3’ end target sequence. Proc Natl Acad Sci USA 1998; 95:4469–4474 [View Article] [PubMed]
     [Google Scholar]
  41. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 2012; 7:1848–1857 [View Article] [PubMed]
     [Google Scholar]
  42. Dixon AS, Schwinn MK, Hall MP, Zimmerman K, Otto P et al. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem Biol 2016; 11:400–408 [View Article] [PubMed]
     [Google Scholar]
  43. Thao TTN, Labroussaa F, Ebert N, Jores J, Thiel V. In-yeast assembly of Coronavirus infectious cDNA clones using a synthetic genomics pipeline. In Maier HJ, Bickerton E. eds Coronaviruses Methods Protoc New York, NY: Springer US; 2020 pp 167–184 [View Article]
     [Google Scholar]
  44. Zhou Y, Li C, Ren C, Hu J, Song C et al. One-step assembly of a porcine epidemic diarrhea virus infectious cDNA clone by homologous recombination in yeast: rapid manipulation of viral genome with CRISPR/Cas9 gene-editing technology. Front Microbiol 2022; 13: [View Article]
     [Google Scholar]
  45. Huang J, Brieba LG, Sousa R. Misincorporation by wild-type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation. Biochem 2000; 39:11571–11580 [View Article] [PubMed]
     [Google Scholar]
  46. Almazán F, González JM, Pénzes Z, Izeta A, Calvo E et al. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 2000; 97:5516–5521 [View Article] [PubMed]
     [Google Scholar]
  47. Yount B, Curtis KM, Fritz EA, Hensley LE, Jahrling PB et al. Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 2003; 100:12995–13000 [View Article] [PubMed]
     [Google Scholar]
  48. Xie X, Muruato A, Lokugamage KG, Narayanan K, Zhang X et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 2020; 27:841–848 [View Article] [PubMed]
     [Google Scholar]
  49. Donaldson EF, Yount B, Sims AC, Burkett S, Pickles RJ et al. Systematic assembly of a full-length infectious clone of human coronavirus NL63. J Virol 2008; 82:11948–11957 [View Article] [PubMed]
     [Google Scholar]
  50. Scobey T, Yount BL, Sims AC, Donaldson EF, Agnihothram SS et al. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. Proc Natl Acad Sci USA 2013; 110:16157–16162 [View Article] [PubMed]
     [Google Scholar]
  51. Diefenbacher MV, Baric TJ, Martinez DR, Baric RS, Catanzaro NJ et al. A nano-luciferase expressing human coronavirus OC43 for countermeasure development. Virus Res 2024; 339:199286 [View Article] [PubMed]
     [Google Scholar]
  52. Thiel V, Siddell SG. Reverse genetics of coronaviruses using vaccinia virus vectors. In Coronavirus Replication Reverse Genet 2005 pp 199–227 [View Article] [PubMed]
     [Google Scholar]
  53. Almazán F, DeDiego ML, Sola I, Zuñiga S, Nieto-Torres JL et al. Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate. mBio 2013; 4:e00650-13 [View Article] [PubMed]
     [Google Scholar]
  54. Torii S, Ono C, Suzuki R, Morioka Y, Anzai I et al. Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction. Cell Rep 2021; 35:109014 [View Article] [PubMed]
     [Google Scholar]
  55. Kipfer ET, Hauser D, Lett MJ, Otte F, Urda L et al. Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform. Elife 2023; 12:RP89035 [View Article] [PubMed]
     [Google Scholar]
  56. Ye C, Chiem K, Park J-G, Oladunni F, Platt RN 2nd et al. Rescue of SARS-CoV-2 from a single bacterial artificial chromosome. mBio 2020; 11:e02168-20 [View Article] [PubMed]
     [Google Scholar]
  57. Fahnøe U, Pham LV, Fernandez-Antunez C, Costa R, Rivera-Rangel LR et al. Versatile SARS-CoV-2 reverse-genetics systems for the study of antiviral resistance and replication. Viruses 2022; 14:172 [View Article] [PubMed]
     [Google Scholar]
  58. Mélade J, Piorkowski G, Touret F, Fourié T, Driouich J-S et al. A simple reverse genetics method to generate recombinant coronaviruses. EMBO Rep 2022; 23:e53820 [View Article] [PubMed]
     [Google Scholar]
  59. Aubry F, Nougairède A, de Fabritus L, Piorkowski G, Gould EA et al. “ISA-Lation” of single-stranded positive-sense RNA viruses from non-infectious clinical/animal samples. PLoS ONE 2015; 10:e0138703 [View Article] [PubMed]
     [Google Scholar]
  60. Fehr AR. Bacterial artificial chromosome-based lambda red recombination with the I-sceI homing endonuclease for genetic alteration of MERS-CoV. In Vijay R. eds MERS Coronavirus Methods Protoc New York, NY: Springer US; 2020 pp 53–68 [View Article]
     [Google Scholar]
  61. Freeman MC, Graham RL, Lu X, Peek CT, Denison MR. Coronavirus replicase-reporter fusions provide quantitative analysis of replication and replication complex formation. J Virol 2014; 88:5319–5327 [View Article] [PubMed]
     [Google Scholar]
  62. Bosch BJ, de Haan CAM, Rottier PJM. Coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent. J Virol 2004; 78:7369–7378 [View Article] [PubMed]
     [Google Scholar]
  63. Burkard C, Bloyet L-M, Wicht O, van Kuppeveld FJ, Rottier PJM et al. Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of β-galactosidase. PLoS One 2014; 9:e101762 [View Article] [PubMed]
     [Google Scholar]
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A yeast-assembled, plasmid-launched reverse genetics system for the murine coronavirus MHV-A59
J. Gen. Virol. 106, 002065 (2025); https://doi.org/10.1099/jgv.0.002065
/content/journal/jgv/10.1099/jgv.0.002065
/content/journal/jgv/10.1099/jgv.0.002065
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