📄 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 (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. © 1992 IRL Press at Oxford University Press.

keywords 🔗 defective interfering (26) 🔗 virus replication (219) 🔗 viral genome (96) 🔗 infected cells (307)

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