Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups

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 ts 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 ts 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 ts 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.