Functional Analysis of the Herpes Simplex Virus Polypeptide Vmw65

Ramsay, Fiona Helen (1987) Functional Analysis of the Herpes Simplex Virus Polypeptide Vmw65. PhD thesis, University of Glasgow.

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Abstract

The aim of this project was to study the role of Vmw65, a virion tegument polypeptide, which is important not only as a structural protein, but also as the trans-inducing factor (TIF) of HSV immediate-early (IE) gene transcription. To elucidate the role of Vmw65 in the assembly of infectious virions, the HSV-2 mutant, ts2203, derived from the multiple mutant, tsl3, was characterised. Previous work strongly suggested that tsl3 has a temperature sensitive (ts) lesion within the Vmw65 gene, since Vmw65 produced by tsl3 failed to react with the monoclonal antibody, MA1044, directed against this polypeptide. In contrast, the wild type (WT) HG52 HSV-2 Vmw65, or the gene product synthesised by ts revertants of tsl3, which retained the defect within the alkaline exonuclease gene, was immunoprecipitated by the antibody (H. M. Moss and J. W. Palfreyman, personal communication). Preliminary studies on tsl3 indicated that the virus has a late defect and therefore it was likely that the lesion affected the structural function of Vmw65. To obtain a mutant with a single ts lesion, a cloned fragment, BglII i, containing the tsl3 Vmw65 gene, was recombined into WT HSV-2 strain HG52, and the mutant, ts2203, isolated. The ability of the plasmid pMCl, which contains the HSV-1 Vmw65 gene, to rescue ts2203 supported the idea that ts2203 had a lesion within Vmw65. Results from marker rescue experiments using HSV-2 HG52 cloned DNA fragments, together with information from cross-hybridisation studies with HSV-1 pMCl and subfragments of HSV-2 BglII i, suggested that the mutation was present at the 5' end of the coding sequences. The isolation of ts revertants which formed plaques at the NPT with the same efficiency as WT virus confirmed that this mutant had a single ts lesion. Polypeptide analysis of virus-infected cell extracts showed that ts2203 synthesised at both the PT and the NPT, a Vmw65 which had an altered electrophoretic mobility on SDS polyacrylamide gels. Two of five ts revertants of ts2203 also specified a polypeptide with this property whilst the remainder induced Vmw65 which had a similar mobility to WT virus. This information suggests that the ts lesion in the Vmw65 gene is responsible for the alteration in the electrophoretic mobility of Vmw65 encoded by ts2203. Since ts2203 BglII i cloned DNA retained the ability to stimulate IE transcription to the same extent as WT HG52 BglII i, the lesion in ts2203 must only affect the structural function of Vmw65. To determine which stage of virion assembly was affected by the lesion, thin section preparations of ts2203- or WT HG52-infected cells grown at the PT and NPT were examined under the electron microscope. Similar numbers of capsids were present in the nuclei of cells infected by either virus at the NPT, suggesting that ts2203 did not have a defect in the assembly of capsids. At this temperature, however, WT virus produced low numbers of dense cored capsids, making it difficult to ascertain whether there was any difference in the number of DNA-containing capsids in the mutant-infected cells compared with those cells infected with WT virus. Analysis of viral DNA synthesised at the NPT revealed that most of the viral DNA was endless, supporting the electron microscopic observations that both viruses packaged DNA inefficiently at this temperature. These results together with the finding that cells infected with HG52 at the NPT contained very few enveloped particles, presumably because little of the replicated concatemeric viral DNA was cleaved and encapsidated, meant that it was impossible to determine whether the ts2203 defect was affecting packaging of viral DNA or envelopment of DNA-containing capsids. To overcome this problem, the BglII i fragment of tsl3 was recombined into another HSV-2 strain, HVD25766, which grows well at the NPT and, in contrast to strain HG52, produces large amounts of cell-released enveloped virus. The resulting HVD mutant, ts2204, was compared with WT HVD for the ability to encapsidate virus DNA at the PT and NPT, and initial results indicated that the mutant had a packaging defect. However, a ts virus, produced by recombining cloned HG52 BglII i into ts2204 DNA, also failed to package significant amounts of viral DNA at the NPT. It was, therefore, concluded that ts2204 had more than one ts mutation. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-77567
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53
URI: http://theses.gla.ac.uk/id/eprint/77567

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