Site-directed mutagenesis of the genome of mouse hepatitis virus by targeted RNA recombination

We have genetically characterized a nucleocapsid (N) protein mutant of the coronavirus mouse hepatitis virus (MHV). This mutant, designated Alb4, is both temperature-sensitive and thermolabile, and its N protein is smaller than wild-type N. Sequence analysis of the Alb4 N gene revealed that it contains an internal deletion of 87 nucleotides, producing an in-frame deletion of 29 amino acids. All of these properties of Alb4 made it ideal for use as a recipient in a targeted RNA recombination experiment in which the deletion in Alb4 was repaired by recombination with synthetic RNA7, the smallest MHV subgenomic mRNA. Progeny from a cotransfection of Alb4 genomic RNA and synthetic RNA7 were selected for thermal stability. PCR analysis of candidate recombinants showed that they had regained the material that is deleted in the Alb4 mutant. They also had acquired a five nucleotide insertion in the 3' untranslated region, which had been incorporated into the synthetic RNA7 as a molecular tag. The presence of the tag was directly verified, as well, by sequencing the genomic RNA of purified recombinant viruses. This provided a clear genetic proof that the Alb4 phenotype was due to the observed deletion in the N gene. In addition, these results demonstrated that it is possible to obtain stable, independently replicating progeny from recombination between coronaviral genomic RNA and a tailored, synthetic RNA species. To date, we have constructed three additional mutants by this procedure. For two of these, a second-site point mutation that reverts the Alb4 phenotype has been transduced into a wild type background, which does not contain the Alb4 deletion.(ABSTRACT TRUNCATED AT 250 WORDS)