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Title: The control of herpes simplex virus late gene transcription
Author: Johnson, Paul Andrew
ISNI:       0000 0001 3591 3385
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1987
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The coordinate temporal control of the HSV-1 transcriptional programme involves three main phases of gene expression; immediate-early (IE), early (E), and late (L). The features which distinguish IE genes are well defined. IE genes are transcribed in the absence of de novo protein synthesis. Their transcription is stimulated by a component of the virus particle, Vmw65, and this requires the presence of a "far-upstream region" which includes one or more copies of the consensus sequence, 5'-TAATGARATTC-3'. IE gene products are required for the activation of the later classes of viral genes, whose expression in the absence of DNA replication is either maximal (E genes), sub-maximal (EL genes) or very poor (L genes). The organization of the promoter sequences of IE and E genes resembles that of many other eukaryotic (non-"housekeeping") genes, characterized by (i) a proximal element bearing a TATA box homology, and (ii) distal elements which are recognized by cellular transcription factors such as Spl and CTF. E genes do not possess far-upstream regulatory elements. Until recently, very little was known about late gene expression: What distinguishes a late promoter from an early promoter; why is efficient late gene expression dependent on DNA replication? The work in this thesis has been directed at the control of expression of an HSV-1 late gene, US11. It was shown that the US11 gene was regulated in the virus with "true-late" kinetics, in terms of the time-course of appearance of US11 RNA and the sensitivity of US11 gene expression to inhibition of DNA replication. Under conditions of DNA replication inhibition, the accumulation of US11 RNA was reduced by 50 to 100-fold. In contrast, the accumulation of RNA from an EL gene, glycoprotein D (gD), was reduced by only 5 to 10-fold. Thus there is a clear difference between the regulation of transcription of gD, an EL gene, and US11, a "true-late" gene. A plasmid system was developed in order to study the regulation of the US11 promoter. Plasmid DNA was introduced into tissue culture cells by a short-term transfection procedure, followed by infection with virus to provide the necessary factors for activation of US11. The US11 promoter was therefore linked to the coding portion of the rabbit beta-globin gene, in order to distinguish between transcripts derived from the plasmid and those from the virus. The activity of the plasmid-borne US11 promoter in constructs containing or lacking a functional HSV-1 origin of DNA replication (ORIs) was analysed by quantitative SI mapping of correctly initiated hybrid transcripts. Following HSV-1 infection of transfected HeLa cells, the US11 promoter in ORI+ plasmids was expressed with similar kinetics to its viral counterpart. US11 promoter activity was first detected at the same time as the onset of DNA template replication. Expression of US11 RNA was detectable from non-replicating ORI- plasmids, although transcript accumulation was reduced by greater than 90%. Sequences containing the IE gene 5 promoter (a 3' co-terminal gene whose transcription starts 5' of US11) also played a positive role in achieving normal US11 gene expression, which suggested that both replication and through-transcription may act by inducing structural changes to the late promoter DNA. The DNA sequence requirements for US11 gene expression were investigated using plasmids which contain ORIs. The sequences necessary for fully efficient regulated expression of US11 lie within the region -31 to +39 relative to the RNA start sites. It appeared that a late promoter might consist only of a proximal TATA box-cap site region. This hypothesis was tested by removing the distal upstream region of the gD promoter (which is required for its normal regulation as an EL promoter) and linking this truncated promoter to ORIs. The result was the conversion of gD promoter regulation to late gene kinetics during virus super infection. It therefore seems unlikely that late promoters are distinguished by late promoter-specific sequences. Co-transfection of the US11 promoter with plasmids bearing HSV-1 IE genes showed that US11 could be activated by Vmw175 and Vmw110 and most efficiently, by the two in combination. This suggests that the target sequence for trans-activation by HSV 1E gene products may be in the TATA box region. These results are discussed in terms of the possible mechanisms of regulation of late genes, and of transactivation of transcription in general, and also in comparison with late gene regulation in other viral systems.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Microbiology