The Regulation of Herpes Simplex Virus Immediate Early Gene Expression

The experiments presented in this thesis deal with two aspects of the transcriptional control of herpes simplex virus type 1 (HSV-1) immediate early (IE) gene expression. Stimulation of the IE gene set is mediated by a protein component of the virion, Vmw65, whose gene has been mapped on the viral genome (Campbell et al, 1984). A specific DNA fragment, BamHI F, containing the entire coding and flanking sequences for Vmw65 has been sequenced using a bacteriophage M13 shot-gun cloning strategy and the dideoxy sequencing technology. The fragment is 8,055 base pairs in size. The mRNA for Vmw65 has been positioned precisely, by nuclease SI mapping. The predicted open reading frame for Vmw65 consists of 490 codons and translates to a protein of molecular weight 54,342. The protein would appear to have no outstanding physical characteristics, such as regions of extreme hydrophobicity or hydrophilicity, but has significantly more acidic residues than the average protein, especially towards the carboxy terminus. The 5' and 3' flanking sequences of the gene exhibit recognisable signals known to be involved in the regulation of transcription and termination of eukaryotic genes. A homologue to Vmw65 has been identified in the genome of varicella-zoster virus (VZV; A. J. Davison, personal communication). The proteins from the two viruses are highly conserved at the level of amino acid sequence and approximately colinear, although the VZV equivalent is 80 amino acids shorter than Vmw65 at the carboxy terminus. Three other genes can be detected in the sequence of BamHI F, consistent with the mRNA mapping of Hall et al (1982). The functions of the products of these genes are unknown. All three genes have homology to genes on the VZV genome at the level of amino acid sequence, but the degree of conservation is variable. The region of VZV which is analogous to the BamHI F fragment shows an identical arrangement of genes to HSV-1. The second aspect of IE gene control which has been investigated is the reported autoregulation of IE gene 3 expression by its product Vmw175. This is based on experiments which showed Vmw175 to be present in lower abundance at early and late times, when compared to IE times, and the observation that a temperature sensitive mutant in Vmw175 (tsK) overproduces IE gene products. The investigation involved the use of plasmid constructs in which the HSV-1 thymidine kinase (TK) was placed under the control of IE promoters. These plasmids were analysed for expression of TK activity after introduction into tissue culture cells, together with the cloned Vmw175 gene from wild type or tsK virus, by calcium-phosphate transfection. The results presented confirm that autoregulation does occur. Polypeptide Vmw175 is able to stimulate expression from the promoters of IE genes 2 and 4/5, but not from its own promoter. This finding suggests that autoregulation may occur indirectly via competitive exclusion of the IE gene 3 promoter from the transcription machinery at post-immediate early times. The reason why Vmw175 cannot activate its own promoter is unclear, however some evidence is presented implicating the enhancer region of IE gene 3. The observation that Vmw175 activates other IE promoters, presumably in an analogous manner to the activation of early and late gene expression, may provide an insight into the role of these IE gene products in the viral lytic cycle