Isolation and Characterization of Conditional Lethal Amber Nonsense Mutants of Vesicular Stomatitis Virus

White, Brian Templeton (1987) Isolation and Characterization of Conditional Lethal Amber Nonsense Mutants of Vesicular Stomatitis Virus. PhD thesis, University of Glasgow.

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The work reported in this thesis is concerned with the isolation and characterization of conditional lethal amber nonsense mutants of the rhabdovirus vesicular stomatitis virus (VSV), Indiana serotype. Two murine cell lines were employed in this work. L39 (su+) cells carried an amber suppressor tRNA gene, which was expressed, albeit at a relatively low level. These cells thus represented the permissive host for virus containing a conditional lethal amber mutation. The second cell line used was the cell line LMTK- (su-). This was the parental line from which the L39 cell line was derived, and represented the non-permissive host for virus containing a nonsense mutation in an essential gene. A screen was set up to detect conditional lethal nonsense mutants of VSV, by assaying growth of mutagenized isolates on su+ and su- cells and retaining those which grew only on su+ cells. A wt stock of VSV (Indiana serotype) was mutagenized by the chemical mutagen 5-fluorouracil. The mutagenized virus was plated on the su+ cells to produce well isolated plaques. Small plaques were picked to screen for a mutation affecting host range. Screening was performed by infecting monolayers of su~ cells and inspecting the monolayers for the presence or absence of cpe. Any isolates which did not give rise to cpe were subsequently used to infect monolayers of su+ cells. The virus from productive infections was harvested and titrated on both cell lines to quantify the restriction in host range. Of 2614 plaques picked originally, 82 did not give rise to cpe on the su- cells and of these 13 produced a difference in titre between the two cell lines of 30 fold or more. Viral protein synthesis in su+ and su- cells infected by each of these host range mutants was investigated by polyacrylamide gel electrophoresis. In comparison to the characteristic protein species synthesized by wt virus, seven mutants exhibited little virus specific protein synthesis in su" cells. Complementation analysis was carried out for each of these seven mutants with ts mutants representing each of the five VSV complementation groups. Five of the mutants were assigned to complementation group I (the L cistron), one to complementation group V (the G cistron) while a seventh mutant could not be complemented. No intragenic complementation was observed between any of the group I mutants. The five group I mutants (retrospectively named AmbLl to AmbL5) did not synthesize any detectable full length L protein in the su+ cells. Four (AmbLl to AmbL4) synthesized an additional protein species in the su+ cells. An additional protein could not be identified for the fifth mutant, however. The molecular weights of the additional proteins were estimated to be 37000, 150000, 230000 and 227000 for AmbLl, AmbL2, AmbL3 and AmbL4 respectively (full length L protein has a molecular weight of 241012). The additional proteins were also detected by in vitro translation of mRNA isolated from su+ cells infected by the mutants AmbLl and AmbL2. These proteins were proposed to be fragments of the L protein produced by premature termination of translation within the L mRNA at an amber codon. The mutants were found to be greatly restricted in the su- cells. Analysis of the proteins synthesized in su-cells revealed that the inhibition of host protein synthesis associated with wt infection of su- cells was markedly reduced. The additional protein synthesized by these mutants in su+ cells was only observed in su- cells with some of the mutants (AmbLl, AmbL3 and AmbL4) after prolonged incubation. This is thought to be caused by outgrowth of revertants complementing the mutant late in infection. Analysis of the protein composition of the virion of these mutants showed that the shortened form of the L protein was not packaged. The additional protein synthesized by AmbLl and AmbL2 (and possibly AmbL4) in the su+ cells was present in considerably higher abundance than either wild-type L protein or the truncated L protein found in AmbL3 infected cells. This is thought to reflect the action of a control system on L protein synthesis. Immunoprecipitation and immunoblotting analysis of the group I mutants with monospecific antisera directed against the N and C termini of the wt L protein provided direct evidence that the additional proteins produced by the group I mutants were derivatives of L protein and possessed N-terminal specific sequences but not C-terminal specific sequences. Immunoprecipitation and immunoblotting studies with AmbL5 each revealed a possible (but distinct) candidate for an L truncation protein. These results reinforce the interpretation that the mutants synthesize a truncated form of the L protein in the su+ cells as a result of premature termination at an amber nonsense codon within the L mRNA. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-77583
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 09:04
Last Modified: 14 Jan 2020 09:04

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