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

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.