Study of the transcription and the replication of vesicular stomatitis virus by using temperature-sensitive mutants

Abstract

The viral ribonucleic acids (RNA species) synthesized in HeLa cells infected with temperature-sensitive (ts) mutants and with the wild type of vesicular stomatitis virus at permissive (30 C) and nonpermissive (39.2 C) temperatures were compared by sucrose gradient centrifugation. Two ts mutants (ts 5 and ts 100) representing two separate complementation groups (respectively, groups I and IV), each concerned with viral RNA synthesis, were chosen. Mutant ts 5 failed to synthesize any RNA at 39.2 C. Under the same conditions, mutant ts 100 showed a low, but easily detectable, synthesis of RNA without characteristic peaks. The in vitro transcriptase activity was tested with mutants ts 5 and ts 100 at 39.2 C: normal activity, compared with wild-type virus, was detected with purified ts 100, but no activity was detected with purified ts 5. From all our data we conclude that mutant ts 5 is defective in transcription. The defect could be in the structural transcriptase enzyme or at the level of template for transcription. Results with mutant ts 100 were not so clear-cut. However, we suggest that this mutant is concerned with some aspect of transcription in vivo. In addition, our results lead us to postulate some linkage between transcription and replication.