Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796249
Title: Construction and characterisation of a herpes simplex virus mutant deficient for Vmw65-mediated stimulation of immediate early gene transcription
Author: Ace, Christopher Ian
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1989
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Abstract:
Herpes simplex virus (HSV) gene expression can be classified into three phases, immediate early (IE), early and late. The 5 IE genes are the first to be transcribed after infection and the products (IE polypeptides) are essential for early and late gene expression. A distinctive feature of HSV gene activation is the stimulation of IE transcription by the late polypeptide Vtnw65, the major tegument protein. The transinduction of IE transcription by Vtnw65 is dependent on the cis- acting regulatory element TAATGARAT which is located in the promoters of all IE genes. Polypeptide Vmw65 interacts with cell factors, including the "octamer"-binding transcription factor, OCT-1, to form a complex (named IEC) which binds specifically to DNA sequences containing TAATGARAT and it is thought that IEC is an important mediator of IE gene transinduction. Thus, Vtnw65 is involved in initiation of virus gene expression at a very early stage of infection. In addition to its role as an IE gene activator, Vmw65 is essential for virion assembly during the latter stages of infection and as a result large scale insertions or deletions in the polypeptide are not tolerated by the virus. The aim of the project was to investigate, by in vitro mutagenesis, the importance and relevance of Vmw65 as an IE gene transinducer in viral infection. The approach taken initially was to analyse the effect of small oligonucleotide insertions in cloned fragments of the gene, in an attempt to map domains within the polypeptide important for IE transinduction and for virion assembly. A series of ten plasmids each with a 12 base pair, BamHI linker, insertion in the gene encoding vmw65 of HSV-1 was constructed. The plasmids were analysed for their transinducing activity in transient cotransfection experiments, and mutant polypeptides synthesised in vitro were analysed for their ability to form IEC in gel retardation experiments. The ability of mutant polypeptides to be correctly assembled into virus particles was assayed by the insertion plasmids' ability to marker rescue a ts virion assembly defective mutant of HSV-2 whose lesion resides in the Vmw65 homologue and which does not affect the transinducing function. It was found that approximately 50% of the insertions abolished the ability of Vmw65 to transinduce IE promoters and that those polypeptides defective in activity did not form IEC, and vice versa. This striking correlation between complex formation and stimulation of transcription is convincing evidence that IEC is an essential intermediate of IE transinduction. When the mutant plasmids were analysed for their virion assembly phenotype, again approximately 50% were non-functional but the regions important for virion assembly mapped to regions distinct from those required for transinduction. One of the plasmid mutations which abolished transinduction but which had a wild-type (wt) virion assembly phenotype was introduced into virus by intra-typic recombination. A virus, named in1814, containing the linker mutation was isolated and its DNA characterised by Southern blotting. A distinctive feature of in1814 was its reduced ability to grow and produce plaques in tissue culture cells at low multiplicities of infection (moi). The plaquing efficiency of in 1814 was cell-type dependent and was reduced by 100-fold in BHK and Vero cells and as much as 10,000-fold in HFL cells. The mutant virus produced equivalent numbers of virion particles as wt HSV-1 stocks and analysis of virus adsorption to cells and DNA migration to the nucleus showed that in 1814 was able to enter cells efficiently and that the infectivity of the virus particles was normal. Therefore the impaired growth of in 1814 is unlikely to result from -a defect in virion structure. A "revertant" virus was constructed by marker rescue of in 1814 with a DNA fragment containing a wt coding sequence of Vw65. This rescued virus behaved as wt HSV-1, indicating that the phenotype of in1814 does not result from a second site mutation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796249  DOI: Not available
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