Affiliation:
1. Medical Research Council Virology Unit, Institute of Virology, University of Glasgow, Glasgow G11 5JR, Scotland
Abstract
The virion-associated RNA transcriptase activity of vesicular stomatitis virus New Jersey temperature-sensitive (
ts
) mutants was assayed in vitro at the permissive (31°C) and restrictive (39°C) temperatures. RNA synthesis at 39°C by the RNA-negative
ts
A1 and the RNA-positive
ts
C1 and
ts
D1 mutants was similar to that of wild-type virus. The RNA-negative
ts
B1 synthesized only small amounts of RNA in vitro at 39°C. The three mutants of complementation group E were dissimilar in the amounts of RNA they synthesized at 39°C:
ts
E1 synthesized very little RNA,
ts
E2 synthesized moderate amounts, and RNA synthesis by
ts
E3 was not inhibited. The two mutants of group F were also dissimilar, since
ts
F1 synthesized very little RNA at 39°C, whereas
ts
F2 synthesized as much RNA as wild-type virus. The revertant clones
ts
B1/R1,
ts
E1/R1, and
ts
F1/R1 synthesized RNA at 39°C in amounts comparable to wild-type virus, indicating that the heat sensitivity of the transcriptase activity of the mutants
ts
B1,
ts
E1, and
ts
F1 was associated with temperature sensitivity. Similar heat sensitivities were observed when transcribing nucleoprotein complexes were used in the assays, showing that the mutated polypeptides were part of the viral core. The heat stability of the mutant
ts
B1 was similar to that of wild-type virus, and in vitro RNA synthesis was fully restored when the temperature was lowered to 31°C after 30 min of preincubation at 39°C, showing that the inhibition was due to reversible configurational change of the mutated polypeptide. When virions of the mutant
ts
E1 were heated for 5 h at 39°C, their infectivity and transcriptase activity were as stable as those of the wild-type virus, whereas transcriptase activity became very heat labile after disruption of the viral coat with a neutral detergent. This suggests an interaction between the mutated polypeptide and a coat polypeptide which stabilizes the activity of the transcriptase. The RNA transcriptase activity of the mutant
ts
F1 was also heat labile, although to a lesser extent than that of
ts
E1. Thus, the defects in transcriptase activity of groups B, E, and F suggest that all three polypeptides of the virus core, polypeptides L, N, and NS, are involved in the transcription. In addition, we postulate that the mutated gene products of groups E and F are multifunctional, being required both in transcription and replication, and that the gene product of group E may also be involved in some late stage of virus development.
Publisher
American Society for Microbiology
Subject
Virology,Insect Science,Immunology,Microbiology
Cited by
12 articles.
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