Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs.
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. The construction of a full-length cDNA clone, pMIDI, of a DI 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 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.
van der Most
R G
RG
Department of Virology, Academic Hospital Leiden, The Netherlands.
Heijnen
L
L
Spaan
W J
WJ
de Groot
R J
RJ
D001483:Base Sequence; D002460:Cell Line; D003332:Coronaviridae / Q000235:genetics*; D003673:Defective Viruses / Q000235:genetics*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009843:Oligoribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D009843:Oligoribonucleotides; D012367:RNA, Viral