Characteristics of single- and double-stranded RNA synthesis by a rotavirus SA-11 mutant thermosensitive in the RNA polymerase gene.

Abstract

The phenotype of a rotavirus SA-11 mutant, ts C, which carries a mutation in the gene coding for the viral RNA polymerase was studied in vitro. ts C viral transcription proved to be sensitive to temperature in a different way to that previously described. Like the wild type, the ts C mutant has an optimum for in vitro transcription at 45 degrees, but mRNA synthesis was inhibited at temperatures over 50 degrees. This mutant also showed a higher resistance to transcriptional inhibition by nucleotide analogues than the wild-type strain. The in vitro minus-strand RNA synthesis catalysed by ts C particles indicates that the mutant does not exhibit the expected increased sensitivity to temperatures over 31 degrees shown by the in vivo phenotype. As with plus-strand synthesis, the optimal temperature for the minus-strand synthesis assay was 45 degrees, but for temperatures over 55 degrees, the number of double-stranded RNA products was altered. Our results suggest that when in vitro plus- and minus-strand RNA synthesis in ts C and wild type are compared, the mutated VP1 motif affects both transcription and minus-strand synthesis, but in different ways. In infected cell cultures, the results also show that the phenotype associated with ts C seems to mainly affect the function of plus-strand RNA synthesis.