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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.
Insights
Researchers identified temperature-dependent host range mutants in vesicular stomatitis virus. These mutants reveal a heat-sensitive RNA transcriptase essential for viral replication, impacting virus assembly.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Vesicular stomatitis virus (VSV), New Jersey serotype, is a model for studying RNA virus replication.
- Conditional lethal mutants are crucial tools for dissecting viral gene function.
- 5-fluorouracil is a chemical mutagen used to generate viral mutants.
Purpose of the Study:
- To characterize conditional lethal mutants of VSV, New Jersey serotype.
- To investigate the role of virion-associated RNA transcriptase in host range and temperature sensitivity.
- To identify mutations affecting viral RNA synthesis.
Main Methods:
- Isolation and characterization of temperature-sensitive (ts) and host range (hr, td) mutants of VSV.
- Assay of virion-associated RNA transcriptase activity in vitro at different temperatures (31°C and 39°C).
- Comparison of enzyme activity between wild-type, mutant, and revertant viruses.
Main Results:
- Three classes of mutants were isolated: ts, hr CE, and temperature-dependent host range (td CE) mutants.
- Host range mutants (hr CE) showed no alteration in RNA transcriptase activity.
- Temperature-dependent host range mutants (td CE) were divided into two groups; one group exhibited heat-sensitive RNA transcriptase activity.
- Mutant td CE 3 displayed temperature-sensitive transcriptase activity, reversible upon temperature shift.
- Revertant of td CE 3 regained normal RNA synthesis at elevated temperatures.
Conclusions:
- A mutation in the second class of td CE mutants confers temperature sensitivity to a polypeptide involved in the RNA transcriptase complex.
- The virion-associated RNA transcriptase is a likely target of the mutation in td CE mutants.
- The heat sensitivity of the transcriptase in mutant td CE 3 is rapidly reversible, suggesting a direct impact on enzyme function.