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Mouse hepatitis virus has been shown to undergo RNA recombination at high frequency during mixed infection. Temperature-sensitive mutants were isolated using 5-fluorouracil and 5-azacytidine as mutagen. Six RNA+ mutants that reside within a single complementation group mapping within the S glycoprotein gene of MHV-A59 were isolated which did not cause syncytium at the restrictive temperature. Using standard genetic techniques, a recombination map was established that indicated that these mutants mapped into two distinct domains designated F1 and F2. These genetic domains may correspond to mutations mapping within the S1 and S2 glycoproteins, respectively, and suggest that both the S1 and S2 domains are important in eliciting the fusogenic activity of the S glycoprotein gene. In addition, assuming that most distal ts alleles map roughly 4.0 kb apart, a recombination frequency of 1% per 575-676 bp was predicted through the S glycoprotein gene. Interestingly, this represents a threefold increase in the recombination frequency as compared to rates predicted through the polymerase region. The increase in the recombination rate was probably not due to recombination events resulting in large deletions or insertions (greater than 50 bp), but rather was probably due to a combination of homologous and nonhomologous recombination. A variety of explanations could account for the increased rates of recombination in the S gene.

MHV-A59 temperature-sensitive mutants, representing one RNA+ and five RNA− complementation groups, were isolated and characterized by genetic recombination techniques. Maximum recombination frequencies occurred under multiplicities of infection greater than 10 each in which 99.99% of the cells were co-infected. Recombination frequencies between different is mutants increased steadily during infection and peaked late in the virus growth cycle. These data suggest that recombination is a late event in the virus replication cycle. Recombination frequencies were also found to range from 63 to 20,000 times higher than the sum of the spontaneous reversion frequencies of each is mutant used in the cross. Utilizing standard genetic recombination techniques, the five RNA− complementation groups of MHV-A59 were arranged into an additive, linear, genetic map located at the 5′ end of the genome in the 23-kb polymerase region. These data indicate that at least five distinct functions are encoded in the MHV polymerase region which function in virus transcription. Moreover, using well-characterized is mutants the recombination frequency for the entire 32-kb MHV genome was found to approach 25% or more. This is the highest recombination frequency described for a nonsegmented, linear, plus-polarity RNA virus.