Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs

  • R. de Groot
  • L. Heijnen
  • R. van der Most
  • W. Spaan
Conference paper
Part of the Archives of Virology Supplementum book series (ARCHIVES SUPPL, volume 9)


We describe a novel strategy to site-specifically mutagenize the genome of an RNA virus by exploiting homologous RNA recombination between synthetic defective interfering (DI) RNA and viral RNA. Marker mutations introduced in the DI RNA were replaced by the wild-type residues during replication. More importantly, however, these genetic markers were introduced into the viral genome; even in the absence of positive selection, MHV recombinants were isolated. This finding provides new prospects for the study of coronavirus replication using recombinant DNA techniques. As a first application, we describe the rescue of the temperature sensitive mutant MHV Albany-4 using DI- directed mutagenesis. Possibilities and limitations of this strategy are discussed.


Yellow Fever Virus Mouse Hepatitis Virus Defective Interfere Marker Mutation Defective Interfere Particle 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Ahlquist P, French R, Janda M, Loesch-Fries LS (1984) Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proc Natl Acad Sci USA 81: 7066–7070PubMedCrossRefGoogle Scholar
  2. 2.
    Fichot O, Girard M (1990) An improved method for sequencing of RNA templates. Nucleic Acids Res 18: 6162PubMedCrossRefGoogle Scholar
  3. 3.
    Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 83: 8122–8126PubMedCrossRefGoogle Scholar
  4. 4.
    Jarvis TC, Kirkegaard K (1991) The polymerase in its labyrinth: mechanisms and implications of RNA recombination. Trends Genet 7: 186–191PubMedGoogle Scholar
  5. 5.
    Keck JG, Stohlman SA, Soe LH, Makino S, Lai MMC (1987) Multiple recombination sites at the 5′-end of murine Coronavirus RNA. Virology 156: 331–341PubMedCrossRefGoogle Scholar
  6. 6.
    Keck JG, Soe LH, Makino S, Stohlman SA, Lai MMC (1988) RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2. J Virol 62: 1989–1998PubMedGoogle Scholar
  7. 7.
    Keck JG, Matsushima GK, Makino S, Fleming JO, Vannier DM, Stohlman SA, Lai MMC (1988) In vivo RNA-RNA recombination of Coronavirus in mouse brain. J Virol 62: 1810–1813PubMedGoogle Scholar
  8. 8.
    Kirkegaard K, Baltimore D (1986) The mechanism of RNA recombination in polio virus. Cell 47: 433–443PubMedCrossRefGoogle Scholar
  9. 9.
    Koetzner CA, Parker MM, Ricard CS, Sturman LS, Masters PS (1992) J Virol 66: 1841–1848PubMedGoogle Scholar
  10. 10.
    Lai C-J, Zhao B, Hori H, Bray M (1991) Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus. Proc Natl Acad Sci USA 88: 5139–5143PubMedCrossRefGoogle Scholar
  11. 11.
    Lai MMC, Baric RS, Makino S, Keck JG, Egbert J, Leibowitz JL, Stohlman SA (1985) Recombination between nonsegmented RNA genomes of murine coronaviruses. J Virol 56: 449–456PubMedGoogle Scholar
  12. 12.
    Makino S, Keck JG, Stohlman SA, Lai MMC (1986) High-frequency RNA recombination of murine coronaviruses. J Virol 57:729–737PubMedGoogle Scholar
  13. 13.
    Makino S, Taguchi F, Fujiwara K (1984) Defective interfering particles of mouse hepatitis virus. Virology 133: 9–17PubMedCrossRefGoogle Scholar
  14. 14.
    Makino S, Fleming JO, Keck JG, Stohlman SA, Lai MMC (1987) RNA recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers. Proc Natl Acad Sci USA 84: 6567–6571PubMedCrossRefGoogle Scholar
  15. 15.
    Masters PS, Sturman LS (1990) Background paper: Functions of the Coronavirus nucleocapsid protein. Adv Exp Med Biol 276: 235–238PubMedGoogle Scholar
  16. 16.
    Rice CM, Levis R, Strauss JH, Huang HV (1987) 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 61: 3809–3819PubMedGoogle Scholar
  17. 17.
    Rice CM, Grakoui A, Galler R, Chambers TJ (1989) Transcription of infectious yellow fever virus RNA from full-length cDNA templates produced by in vitro ligation. New Biol 1: 285–296PubMedGoogle Scholar
  18. 18.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  19. 19.
    Van der Most RG, Bredenbeek PJ, Spaan WJM (1991) A domain at the 3′ end of the polymerase gene is essential for encapsidation of Coronavirus defective interfering RNAs. J Virol 65: 3219–3226PubMedGoogle Scholar
  20. 20.
    Van der Most RG, Heijnen L, Spaan WJM, De Groot RJ (1992) Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of Coronavirus MHV-A59 via synthetic coreplicating RNAs. Nucleic Acids Res 20: 3375–3381PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • R. de Groot
    • 1
  • L. Heijnen
    • 1
  • R. van der Most
    • 1
  • W. Spaan
    • 1
  1. 1.Faculty of Medicine, Institute of Medical Microbiology, Department of VirologyLeiden UniversityLeidenThe Netherlands

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