Virion trascriptase activity differences in host range mutants of vesicular stomatitis virus

Abstract

Three types of conditional lethal mutant were isolated from wild-type vesicular stomatitis virus, New Jersey serotype, after mutagenization by 5-fluorouracil: (i) conventional temperature-sensitive (ts) mutants, which form plaques at 31 C but not at 39 C; (ii) conventional host range mutants (hr CE), which grow in BHK but not in secondary chicken embryo cells; and (iii) temperature-dependent host range mutants (td CE), which form plaques both at 31 and 39 C on BHK cells but only at 31 C on chicken embryo cells. To determine whether the mutation in hr CE and td CE mutants affected the virion-associated RNA transcriptase, this enzyme was assayed in vitro at 31 and 39 C, and the results were compared with those obtained for the wild-type virus. The RNA trascriptase activity of hr CE mutants did not appear to be affected by the mutation. The td CE mutants fall into two classes: those that synthesized RNA at 39 C similar to the wild-type virus and those that did not. One mutant of the latter category, td CE 3, had heat-sensitive transcriptase regardless of whether it was grown in BHK or chicken embryo cells. A revertant to the wild-type phenotype isolated from this mutant had regained the ability to synthesize RNA at 39 C. These results strongly suggest that a polypeptide that is either the transcriptase itself or part of the transcriptase complex was made temperature sensitive by the mutation in the second class of td CE mutants. The inhibition of the transcriptase activity of the mutant td CE 3 was fully reversible by lowering the temperature of incubation from 39 to 31 C, and both inhibition and reactivation appeared to be instantaneous.