RNA- temperature-sensitive mutants of vesicular stomatitis virus: L-protein thermosensitivity accounts for transcriptase restriction of group I mutants

The metabolism of viral RNA and proteins has been studied in cells infected with temperature-sensitive mutant strains of vesicular stomatitis virus. Certain viral proteins encoded by the mutant strains, usually the putative mutant protein for the assigned complementation group, were shown to be degraded more rapidly at the nonpermissive temperature than were the wild-type proteins. Group III mutants (tsG33, tsM301) encode M proteins which are degraded threeto fourfold faster than the wild-type protein. This defect cannot be fully rescued by coinfection with wild-type virus, and thus the defect appears to be in the M protein itself. Mutants tsM601(VI) and tsG41(IV) encode N proteins which are degraded much faster than the wild-type protein and also share the property of being defective in replication of viral RNA, suggesting a correlation between these phenotypic properties. Furthermore, the L proteins of tsGll(I) and tsG13(I) are more labile than the wild-type protein at the nonpermissive temperature. The G protein of tsM501(V) did not undergo the change in electrophoretic mobility previously shown to be the result of sialylation, suggesting that it is defective in maturation or glycosylation at the nonpermissive temperature. Three of the mutants previously isolated in this laboratory, tsM502(V), tsM601(VI), and tsM602(VI), were shown to be defective in viral RNA synthesis at the nonpermissive temperature. Mutant tsM601(VI) was defective mainly in viral RNA replication, whereas tsM502(V) appeared to be totally defective for viral RNA transcription and replication at the nonpermissive temperature.

Section: Fig 1 Pulse-chase Labeling Of Cultures Infected With Wild supporting

confidence: 93%

mentioning

confidence: 89%

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

Knipe¹,

Lodish²,

Baltimore³

1977

“…Cells infected with the mutant tsG114 replicate 42S RNA at the restrictive temperature but do not produce mRNA; upon the addition of cycloheximide, replication is inhibited and transcription is recovered. These results and others suggest that the L protein is part of both the replicative and the transcriptive enzymes (8,15,24).…”

supporting

confidence: 56%

RNA synthesis of vesicular stomatitis virus-infected cells: in vivo regulation of replication

Simonsen¹,

Batt-Humphries²,

Summers³

1979

Pulse-labeling of vesicular stomatitis virus-infected HeLa and BHK cells with [3H]uridine throughout the infectious cycle demonstrated two peaks of uridine incorporation into virus-specific RNA molecules. By separating total RNA synthesis into replication and transcription products, we showed that replication occurs over a shorter period of time in one peak synthesis. The biphasic nature of uridine incorporation is in part due to a general membrane phenomenon of reduced metabolite transport during vesicular stomatis virus infection and in part due to the apparent uncoupling of replication and transcription. A change in the ratio of newly synthesized plus and minus strands of the genome length (42S) RNA was found as the infection proceeded. Early in the infection, plus-stranded 42S RNA comprised 40% of the total genome length RNA synthesis, whereas late in infection, only 15 to 20% of the 42S RNA synthesized was complementary to the virion minus strand. Our data suggest that the rate of synthesis of plus-stranded 42S RNA was constant throughout the infection. The rate of virus release was determined by monitoring the uptake of [3H]uridine into released virus particles. Virus maturation and release are closely associated with the assembly of 42S RNA-containing nucleocapsids.

“…The virus band was removed from the surface of the Flourinert pad and layered onto a preformed linear 0 to 40% sucrose VOL. 27, 1978 gradient containing 1 M NaCl and 1 mM EDTA (7,10). The band of B virions was collected from the gradient and concentrated by centrifugation at 80,000 x g for 90 min.…”

Section: Methodsmentioning

confidence: 99%

Rebinding of transcriptase components (L and NS proteins) to the nucleocapsid template of vesicular stomatitis virus

Mellon¹,

Emerson²

1978

Self Cite

107

The L and NS proteins of vesicular stomatitis virions (New Jersey serotype) were solubilized with Triton X-100 and high-salt buffer and recombined with purified nucleocapsids under conditions similar to those used to reconstitute transcriptase activity in vitro. The nucleocapsid-bound L and NS proteins were separated from unbound proteins on a glycerol gradient. The rebinding of L and NS proteins mimics the in vivo binding in that at saturation the ratio of L and NS molecules to N molecules is approximately the same as observed in the intact virion. L and NS proteins were separated and added back independently and in combination to the template. The purified NS protein bound to the template in the absence of L protein. However, the L protein binding appeared to depend on the presence of NS protein. The presence of Mg2+ and nucleotides, which is required for transcription, was not necessary for the rebinding of L and NS proteins.