Demonstration in vitro of temperature-sensitive elongation of RNA in Sindbis virus mutant ts6

Characterization of conditionally lethal mutants of alphaviruses, Sindbis virus and Semliki Forest virus, has indicated that in almost all the RNA-negative mutants the temperature-sensitive (ts) defect prevents the formation of active transcription complexes at nonpermissive temperature (40 degrees C), but such complexes retain activity at 40 degrees C if formed first at permissive temperature (30 degrees C). Our recent results have extended the characterization of one exception to this finding: Sindbis ts6 transcription complexes, once formed at 30 degrees C, do not function at 40 degrees C. We used an in vitro assay for viral RNA synthesis to determine whether the ts defect was the result of dissociation of the complex or of a failure to elongate RNA chains in a stable complex. Our results indicated that the phenotype of ts6 observed in vivo was retained in vitro. In vivo incorporation into single-stranded 49S and 26S RNA was inhibited simultaneously with its incorporation into replicative intermediates upon shifting ts6-infected cells to 40 degrees C, which was compatible with a defect in elongation. Complexes formed at 30 degrees C and inactivated in vivo by shifting to 40 degrees C were reactivated by incubation in vitro at 30 degrees C but not at 40 degrees C. Thus, the transcription complexes were stable. Nascent RNA chains initiated in vivo and pulse-labeled in vitro were chased into single-stranded 49S and 26S RNA only when incubation was at 30 degrees C, indicating that the ts6 transcription complex was temperature sensitive in elongation. It should be possible to study in vitro other alphavirus RNA-negative mutants that demonstrate a change in viral RNA synthesis after shift to 40 degrees C. These would include ts mutants in the synthesis of subgenomic 26S mRNA and of minus-strand RNA.