Location of the Transcription Defect in Group I Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Abstract

The ribonucleoprotein-dependent RNA transcriptase in vesicular stomatitis B virions of four temperature-sensitive (ts) mutants belonging to complementation group I was analyzed in vitro at permissive (31 C) and restrictive (39 C) temperatures. The RNA-synthesizing activity of all four ts mutants was more labile at 39 C than was the transcriptive activity of wild-type (wt) virions. In order to locate the temperature-sensitive transcription defect in the mutants, wt and ts mutant virions were fractionated by Triton X-100-high salt solubilizer into a sedimentable ribonucleoprotein template and a nonsedimentable enzyme fraction, each of which alone had little or no transcriptive activity. The templateand enzyme-containing fractions of wt virions were then tested for their capacity to restore transcriptive activity at 39 C to corresponding template and enzyme preparations of ts mutant virions. Recombination of' wt template and ts enzymes resulted in no significant restoration of capacity to synthesize RNA at restrictive temperature. In contrast, transcriptive function at 39 C was reconstituted by recombining the wt enzyme with the template component of ts mutants. It appears, therefore, that the enzyme, rather than the template, is the temperature-sensitive component of the transcription complex of group I vesicular stomatitis virus mutants.

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

“…Pulse-chase experiments with tsGll(I) and tsG13(I) revealed that the L protein was lost at 39°C, whereas at 31°C the protein was quite stable. This is consistent with the previous assignment of the defect in group I mutants to the L protein (7,8). The data for the group II mutants are not shown, but we observed no special protein degradation for either tsG21(II) or tsG22(II).…”

“…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). Similar reconstitution studies have led to the suggestion that group IV mutations are defective in the N protein (14).…”

Pathogenicity and Immunogenicity for Mice of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

“…Leakiness of the mutants should not pose a serious problem and, in fact, may be an advantage. Leakiness is probably the explanation for the vaccination efficacy of ts I5 and ts Ill which should have been restricted in protein synthesis because of their defective transcriptase phenotype (11,24). This finding refutes our hypothesis that ts mutants of complementation group I should not serve as vaccines.…”

“…In each case considerably' less virus was required to produce a level of immunity equivalent to that of intraperitoneal vaccination. The somewhat greater protection afforded by ts I5 vaccine may be a function of the leakiness of this mutant (11). In any case the superior immunizing potential of the ts mutants by the intranasal route is presumably attributable to their capacity to synthesize more antigen in susceptible respiratory cells.…”

“…Mutants of at least two of these groups, I and IV, have a phenotype which results in deficient synthesis of ribonucleic acid (RNA-) at nonpermissive temperatures. Although agreement is not universal, ts mutants of group I appear to be defective in transcription (15,24) at the level of virion transcriptase (11) and, therefore, are presumably defective in protein synthesis (17). On the other hand, ts mutants of complementation group IV are probably restricted at the level of ribonucleic acid replication (2a) and should, therefore, be capable of synthesizing proteins at restrictive temperature (30).…”