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.

Analysis of the defects of temperature-sensitive mutants of vesicular stomatitis virus: intracellular degradation of specific viral proteins

“…Temperature-sensitive mutants of vesicular stomatitis virus (VSV) have been isolated in several laboratories and classified into six complementation groups (2,3,6,18,19,21). Group I contains more than one-half of the mutants isolated, and some of these are defective for transcription of mRNA species in vivo at the nonpermissive temperature (15,16,20,23). In addition, reconstitution studies of the transcriptase enzyme showed that the L protein is defective in these mutants (7,8).…”

Temperature-sensitive defect of vesicular stomatitis virus in complementation group II

“…Cells and viruses. All analyses of in vivo RNA syntheses used HeLa cell monolayers grown as described previously (18) or, for experiments in which a low level of residual cellular protein synthesis was required, in chicken embryo cultures (3).…”

“…VSVs (Indiana serotype) investigated were: a heat-resistant wild type with equivalent growth capabilities at 39.5 and 30°C; a ts mutant belonging to complementation group IV, ts IV 0111 (8); a ts mutant belonging to complementation group II, ts II 052 (8); and two independent revertants of wild-type phenotype obtained from subclones of ts II 052. All virus stocks were grown on chicken embryo cells and contained only standard B particles (18). Virus stocks were verified by suitable complementation tests and checked for their inability to grow at 39.5°C or low content of revertants.…”

“…RNA species were isolated from the cytoplasmic extract by addition of sodium dodecyl sulfate (SDS) (1%, wt/vol, final concentration). The RNA species were fractionated at 21,000 rpm for 15 h in the SW27.1 rotor, using a 15 to 30% sucrose gradient in SDS-TEN buffer (18).…”

“…Chemicals. Most compounds were purchased from sources mentioned elsewhere (3,18). Actinomycin D was a gift from Merck, Sharp & Dohme.…”