A Functional Analysis of the Product of Varicella-Zoster Virus Gene 62

Disney, Graham Harvey (1990) A Functional Analysis of the Product of Varicella-Zoster Virus Gene 62. PhD thesis, University of Glasgow.

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In contrast to the increasingly sophisticated knowledge of herpes simplex virus type 1 (HSV-1), our understanding of varicella-zoster virus (VZV) gene regulation is at an early stage. VZV gene 62 is one of a number of VZV genes which, on the basis of homology with known HSV-1 regulatory genes, are predicted to encode gene regulatory polypeptides. The work presented in this thesis investigated the properties and functions of the product of VZV gene 62. VZV gene 62 encodes a polypeptide with a predicted molecular weight of 140,000 (VZV 140k) which shares considerable predicted amino acid sequence homology with the HSV-1 polypeptide, Vmw175 (or ICP4), encoded by IE gene 3. Vmw175 is an essential viral polypeptide with a pivotal role in the activation of early and late viral gene expression, and also repression of immediate early gene expression. There is compelling evidence that VZV 140k is a functional counterpart of Vmw175, and as such may have a critical role in the regulation of VZV gene expression. This hypothesis was tested by inserting VZV gene 62 coding sequences (expressed from the HSV-1 IE-3 promoter) into both IE-3 gene loci in the short region repeats of the HSV-1 genome. The parent virus used for this manipulation was D30EBA, which is a variant of HSV-1 from which the majority of the Vmw175 coding sequences have been deleted. Like other HSV-1 viruses lacking Vmw175 function, D30EBA is able to grow only in cell lines which express Vmw175 constitutively. Significantly, the resulting recombinant virus, HSV-140, is able to propagate (but unable to form obvious plaques) on normal cell lines. The properties of HSV-140 were studied in comparison to HSV-1 by monitoring the time-course of polypeptide expression and DNA replication during normal infection. It was found that HSV-140 synthesises apparently normal amounts of many viral polypeptides during a high multiplicity infection, although the expression of certain late polypeptides (for example, Vmw65, and the major capsid protein) is reduced; this slight defect may be related to the decreased efficiency of HSV-140 DNA replication. At low multiplicity HSV-140 expressed viral polypeptides inefficiently. Surprisingly, the VZV 140K polypeptide was produced in large amounts at later times of a normal infection, indicating that VZV 140k fails to repress the IE-3 promoter. Finally, infection of cells in the presence of actinomycin-D revealed that HSV-140 is much less efficient than wild type virus in the shut-off of host protein synthesis, suggesting that HSV-140 presents reduced amounts of the virion-associated host shut-off factor (encoded by gene UL41). These results indicate that VZV 140K is able to perform most of the functions of Vmw175 during growth of HSV-1, but that differences in detail lead to less efficient virus growth. The properties of VZV 140k have also been studied in transient transfection assays. It was of particular interest to determine whether VZV 140k was able to repress expression from its own promoter (that of gene 62). To this end plasmid pl40CAT was constructed in which VZV gene 62 upstream sequences from -1146 to +57 (which include the complete promoter) were linked to the coding sequences of the chloramphenicol acetyl transferase (CAT) gene. Co-transfection experiments utilizing p140CAT demonstrated that VZV 140k strongly represses expression from its own promoter, thus establishing further functional homology between the polypeptides. Interestingly, Vmw175 is competent in repression of the VZV gene 62 promoter whereas VZV 140k was unable to repress the HSV-1 IE-3 promoter in the reciprocal experiment. The failure of VZV 140k to repress the IE-3 promoter is consistent with observations made with HSV-140 (see above). Plasmids expressing mutant forms of Vmw175 which have small, in-frame insertions into region 2 of the polypeptide (e.g. pI13) are defective in repression of the IE-3 promoter. Similarly, co-transfection of pI13 fails to repress expression of p140CAT. The sequences comprising region 2 of Vmw175 and VZV 140k are among the most highly conserved between the polypeptides. Notably, an analogous plasmid to pI13 which expresses a mutant form of VZV 140k containing an in-frame insertion into homology region 2 (pC34) is likewise impaired in its ability to repress expression of p140CAT. These results strongly suggest that repression of the VZV gene 62 promoter by VZV 140k and Vmw175 involves a similar mechanism to that of repression of the HSV-1 IE-3 promoter by Vmw175. That is, both polypeptides may effect repression of gene 62 by binding to specific sequences at or near the gene 62 capsite.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1990
Depositing User: Enlighten Team
Unique ID: glathesis:1990-78214
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Feb 2020 12:09
Last Modified: 28 Feb 2020 12:09
URI: https://theses.gla.ac.uk/id/eprint/78214

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