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. 1994 Jan;68(1):328–337. doi: 10.1128/jvi.68.1.328-337.1994

Optimization of targeted RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus.

P S Masters 1, C A Koetzner 1, C A Kerr 1, Y Heo 1
PMCID: PMC236292  PMID: 8254744

Abstract

We have recently described a method of introducing site-specific mutations into the genome of the coronavirus mouse hepatitis virus (MHV) by RNA recombination between cotransfected genomic RNA and a synthetic subgenomic mRNA (C. A. Koetzner, M. M. Parker, C. S. Ricard, L. S. Sturman, and P. S. Masters, J. Virol. 66:1841-1848, 1992). By using a thermolabile N protein mutant of MHV (Alb4) as the recipient virus and synthetic RNA7 (the mRNA for the nucleocapsid protein N) as the donor, we selected engineered recombinant viruses as heat-stable progeny resulting from cotransfection. We have now been able to greatly increase the efficiency of targeted recombination in this process by using a synthetic defective interfering (DI) RNA in place of RNA7. The frequency of recombination is sufficiently high that, with Alb4 as the recipient, recombinants can be directly identified without using thermal selection. The synthetic DI RNA has been used to demonstrate that the lesion in another temperature-sensitive and thermolabile MHV mutant, Alb1, maps to the N gene. Sequencing of the Alb1 N gene revealed two closely linked point mutations that fall in a region of the N molecule previously noted as being the most highly conserved region among all of the coronavirus N proteins. Analysis of revertants of the Alb1 mutant revealed that one of the two mutations is critical for the temperature-sensitive phenotype; the second mutation is phenotypically silent.

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Selected References

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  1. Anderson R., Wong F. Membrane and phospholipid binding by murine coronaviral nucleocapsid N protein. Virology. 1993 May;194(1):224–232. doi: 10.1006/viro.1993.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong J., Niemann H., Smeekens S., Rottier P., Warren G. Sequence and topology of a model intracellular membrane protein, E1 glycoprotein, from a coronavirus. Nature. 1984 Apr 19;308(5961):751–752. doi: 10.1038/308751a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banerjee A. K., Rhodes D. P., Gill D. S. Complete nucleotide sequence of the mRNA coding for the N protein of vesicular stomatitis virus (New Jersey serotype). Virology. 1984 Sep;137(2):432–438. doi: 10.1016/0042-6822(84)90237-x. [DOI] [PubMed] [Google Scholar]
  4. Baric R. S., Fu K., Schaad M. C., Stohlman S. A. Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups. Virology. 1990 Aug;177(2):646–656. doi: 10.1016/0042-6822(90)90530-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boursnell M. E., Binns M. M., Foulds I. J., Brown T. D. Sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus. J Gen Virol. 1985 Mar;66(Pt 3):573–580. doi: 10.1099/0022-1317-66-3-573. [DOI] [PubMed] [Google Scholar]
  6. Denison M. R., Perlman S. Translation and processing of mouse hepatitis virus virion RNA in a cell-free system. J Virol. 1986 Oct;60(1):12–18. doi: 10.1128/jvi.60.1.12-18.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fichot O., Girard M. An improved method for sequencing of RNA templates. Nucleic Acids Res. 1990 Oct 25;18(20):6162–6162. doi: 10.1093/nar/18.20.6162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  9. Horsburgh B. C., Brierley I., Brown T. D. Analysis of a 9.6 kb sequence from the 3' end of canine coronavirus genomic RNA. J Gen Virol. 1992 Nov;73(Pt 11):2849–2862. doi: 10.1099/0022-1317-73-11-2849. [DOI] [PubMed] [Google Scholar]
  10. Jeong Y. S., Makino S. Mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic RNA transcription on RNA replication. J Virol. 1992 Jun;66(6):3339–3346. doi: 10.1128/jvi.66.6.3339-3346.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kamahora T., Soe L. H., Lai M. M. Sequence analysis of nucleocapsid gene and leader RNA of human coronavirus OC43. Virus Res. 1989 Jan;12(1):1–9. doi: 10.1016/0168-1702(89)90048-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kapke P. A., Brian D. A. Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. Virology. 1986 May;151(1):41–49. doi: 10.1016/0042-6822(86)90102-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Koetzner C. A., Parker M. M., Ricard C. S., Sturman L. S., Masters P. S. Repair and mutagenesis of the genome of a deletion mutant of the coronavirus mouse hepatitis virus by targeted RNA recombination. J Virol. 1992 Apr;66(4):1841–1848. doi: 10.1128/jvi.66.4.1841-1848.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kunita S., Mori M., Terada E. Sequence analysis of the nucleocapsid protein gene of rat coronavirus SDAV-681. Virology. 1993 Mar;193(1):520–523. doi: 10.1006/viro.1993.1158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lai M. M. Coronavirus: organization, replication and expression of genome. Annu Rev Microbiol. 1990;44:303–333. doi: 10.1146/annurev.mi.44.100190.001511. [DOI] [PubMed] [Google Scholar]
  16. Lai M. M. RNA recombination in animal and plant viruses. Microbiol Rev. 1992 Mar;56(1):61–79. doi: 10.1128/mr.56.1.61-79.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lapps W., Hogue B. G., Brian D. A. Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes. Virology. 1987 Mar;157(1):47–57. doi: 10.1016/0042-6822(87)90312-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liljeström P., Lusa S., Huylebroeck D., Garoff H. In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release. J Virol. 1991 Aug;65(8):4107–4113. doi: 10.1128/jvi.65.8.4107-4113.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Makino S., Taguchi F., Hirano N., Fujiwara K. Analysis of genomic and intracellular viral RNAs of small plaque mutants of mouse hepatitis virus, JHM strain. Virology. 1984 Nov;139(1):138–151. doi: 10.1016/0042-6822(84)90335-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Masters P. S. Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus. Arch Virol. 1992;125(1-4):141–160. doi: 10.1007/BF01309634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pachuk C. J., Bredenbeek P. J., Zoltick P. W., Spaan W. J., Weiss S. R. Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain A59. Virology. 1989 Jul;171(1):141–148. doi: 10.1016/0042-6822(89)90520-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Parker M. M., Masters P. S. Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein. Virology. 1990 Nov;179(1):463–468. doi: 10.1016/0042-6822(90)90316-J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Racaniello V. R., Baltimore D. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science. 1981 Nov 20;214(4523):916–919. doi: 10.1126/science.6272391. [DOI] [PubMed] [Google Scholar]
  25. Rice C. M., Levis R., Strauss J. H., Huang H. V. Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol. 1987 Dec;61(12):3809–3819. doi: 10.1128/jvi.61.12.3809-3819.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sawicki S. G., Sawicki D. L. Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis. J Virol. 1986 Jan;57(1):328–334. doi: 10.1128/jvi.57.1.328-334.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sawicki S. G., Sawicki D. L. Coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in RNA synthesis. J Virol. 1990 Mar;64(3):1050–1056. doi: 10.1128/jvi.64.3.1050-1056.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schreiber S. S., Kamahora T., Lai M. M. Sequence analysis of the nucleocapsid protein gene of human coronavirus 229E. Virology. 1989 Mar;169(1):142–151. doi: 10.1016/0042-6822(89)90050-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Senanayake S. D., Hofmann M. A., Maki J. L., Brian D. A. The nucleocapsid protein gene of bovine coronavirus is bicistronic. J Virol. 1992 Sep;66(9):5277–5283. doi: 10.1128/jvi.66.9.5277-5283.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sethna P. B., Hung S. L., Brian D. A. Coronavirus subgenomic minus-strand RNAs and the potential for mRNA replicons. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5626–5630. doi: 10.1073/pnas.86.14.5626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Soe L. H., Shieh C. K., Baker S. C., Chang M. F., Lai M. M. Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase. J Virol. 1987 Dec;61(12):3968–3976. doi: 10.1128/jvi.61.12.3968-3976.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spaan W., Cavanagh D., Horzinek M. C. Coronaviruses: structure and genome expression. J Gen Virol. 1988 Dec;69(Pt 12):2939–2952. doi: 10.1099/0022-1317-69-12-2939. [DOI] [PubMed] [Google Scholar]
  34. Sturman L. S., Eastwood C., Frana M. F., Duchala C., Baker F., Ricard C. S., Sawicki S. G., Holmes K. V. Temperature-sensitive mutants of MHV-A59. Adv Exp Med Biol. 1987;218:159–168. doi: 10.1007/978-1-4684-1280-2_20. [DOI] [PubMed] [Google Scholar]
  35. Sturman L. S., Holmes K. V. The molecular biology of coronaviruses. Adv Virus Res. 1983;28:35–112. doi: 10.1016/S0065-3527(08)60721-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Vennema H., de Groot R. J., Harbour D. A., Horzinek M. C., Spaan W. J. Primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens. Virology. 1991 Mar;181(1):327–335. doi: 10.1016/0042-6822(91)90499-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Verbeek A., Tijssen P. Sequence analysis of the turkey enteric coronavirus nucleocapsid and membrane protein genes: a close genomic relationship with bovine coronavirus. J Gen Virol. 1991 Jul;72(Pt 7):1659–1666. doi: 10.1099/0022-1317-72-7-1659. [DOI] [PubMed] [Google Scholar]
  38. de Groot R. J., van der Most R. G., Spaan W. J. The fitness of defective interfering murine coronavirus DI-a and its derivatives is decreased by nonsense and frameshift mutations. J Virol. 1992 Oct;66(10):5898–5905. doi: 10.1128/jvi.66.10.5898-5905.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. van der Most R. G., Heijnen L., Spaan W. J., de Groot R. J. 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 Jul 11;20(13):3375–3381. doi: 10.1093/nar/20.13.3375. [DOI] [PMC free article] [PubMed] [Google Scholar]

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