Intracellular RNA synthesis directed by temperature-sensitive mutants of simian rotavirus SA11

Abstract

The kinetics of intracellular synthesis of single-stranded (ss) RNA and double-stranded (ds) RNA directed by prototype temperature-sensitive (ts) mutants representing the 10 mutant groups of rotavirus SA11 were examined. Cells were infected with individual mutants or wild type under one-step growth conditions and maintained at permissive temperature (31 degrees) or nonpermissive temperature (39 degrees). At various times postinfection, infected cells were pulse-labeled, ssRNA and dsRNA were purified, RNA species were resolved by electrophoresis and autoradiography, and RNA synthesis was quantitated by computer-assisted densitometry. The mutants representing all groups synthesized significantly less ssRNA and dsRNA at both 31 degrees and 39 degrees, when compared to wild type. When the ratio of synthesis at 39 degrees/31 degrees was determined for ssRNA and dsRNA of each mutant, three RNA synthesis phenotypes were evident. The tsB(339), tsC(606), and tsE(1400) mutants synthesized both ssRNA and dsRNA in a temperature-dependent manner. The group G mutant, tsG(2130), synthesized ssRNA in temperature-independent fashion but was temperature-dependent for the synthesis of dsRNA. The remaining mutants, tsA(778), tsD(975), tsF(2124), tsH(2384), tsI(2403), and tsJ(2131), synthesized both ssRNA and dsRNA in a temperature-independent fashion. The RNA synthesis phenotypes of the ts mutants are discussed in terms of what is known of the function(s) of the protein species to which ts lesions have been assigned.