Genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates.

Mouse hepatitis virus (MHV) A59 temperature-sensitive (ts) mutants belonging to complementation group C were characterized and mapped by standard genetic recombination techniques. Temperature shift experiments early in infection suggested that the group C allele can be divided into two phenotypically distinct subgroups, designated C1 and C2. Since previous data indicated that the group C1 mutants probably contained an early defect which affects negative-strand synthesis, RNA synthesis was further examined by analyzing replicative-form (RF) RNA. Full-length as well as subgenomic-length RF RNAs were radiolabeled from 3 to 12 h postinfection (p.i.) and labeled late in infection after shift to the nonpermissive temperature (39.5 degrees C). The relative percent molar ratios of each mRNA and corresponding RF RNA were roughly equivalent throughout infection. Temperature shift experiments at 5.5 or 6.0 h p.i. resulted in an 83 to 92% reduction in the amount of total RF RNA at 39.5 degrees C. Radiolabeling time course experiments after temperature shift to 39.5 degrees C also demonstrated incorporation (6 to 9 h p.i.) into both subgenomic-length and full-length RF RNAs, suggesting that previously transcribed negative strands were functional templates throughout infection. To determine if the reduction in RF RNA was due to a decrease in positive- or negative-strand RNA synthesis, rates of mRNA synthesis were calculated from both full-length and subgenomic-length templates. The rate of mRNA synthesis after the shift was increased at 39.5 degrees C compared with that at 32 degrees C regardless of the template used; however, transcription rates calculated from subgenomic-length templates were similar to those of other viral and eukaryotic polymerases. These findings support the notion that the group C1 allele regulates negative-strand RNA synthesis and strongly suggest that the subgenomic negative-strand RNAs are probably the predominant functional templates for the synthesis of positive-strand RNAs late in infection.