Nucleic Acids Research, 1992, Vol. 20, No. 13 3375-3381
© 1992
MOLECULAR BIOLOGY |
Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs
Department of Virology, Institute of Medical Microbiology, Academic Hospital Leiden PO Box 320, 2300 AH Leiden, The Netherlands
* To whom correspondence should be addressed
Received April 3, 1992. Revised June 11, 1992. Accepted June 11, 1992.
We describe a novel strategy to site-specifically mutagenize the genome of an RNA virus by exploiting homologous RNA recombination between synthetic defective interfering (Dl) RNA and the viral RNA. The construction of a full-length cDNA clone, pMIDI, of a Dl RNA of coronavirus MHV strain A59 was reported previously (R.G. Van der Most, P.J. Bredenbeek, and W.J.M. Spaan (1991). J. Virol. 65, 3219–3226). RNA transcribed from this construct, is replicated efficiently in MHV-infected cells. Marker mutations introduced in MIDI 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 Dl-dlrected mutagenesis. Possibilities and limitations of this strategy are discussed.
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