Coronaviruses pp 149-154 | Cite as

Homologous RNA Recombination Allows Efficient Introduction of Site-Specific Mutations into the Genome of Coronavirus MHV-A59 via Synthetic Co-Replicating RNAs

  • Robbert van der Most
  • Leo Heijnen
  • Willy Spaan
  • Raoul de Groot
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 342)


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 the 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 could be 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.


Defective Interfere Marker Mutation Intracellular RNAs Defective Interfere Particle Coronavirus Replication 
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Copyright information

© Springer Science+Business Media New York 1994

Authors and Affiliations

  • Robbert van der Most
    • 1
  • Leo Heijnen
    • 1
  • Willy Spaan
    • 1
  • Raoul de Groot
    • 1
  1. 1.Department of Virology, Institute of Medical Microbiology Faculty of MedicineLeiden UniversityLeidenThe Netherlands

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