Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.795927
Title: The control of herpesvirus immediate early gene expression
Author: Campbell, Moyra Elizabeth Mary
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1987
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Abstract:
The experiments performed in this study investigate the regulation of the immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). Transcription mediated by an HSV-1 IE gene promoter and upstream regulatory sequences is stimulated by a structural virion component (Post et al., 1981). In order to identify the trans-inducing factor (TIF) involved, a series of HSV-1 genomic clones were transfected into baby hamster kidney (BHK) cells together with chimaeric plasmids which contained the thymidine kinase (TK) gene under IE control. Fragments, EcoRI i (0.62 to 0.72 map units), EcoRI b (0.72 to 0.87map units) and BamHI f (0.64 to 0.69 map units), a sub fragment of EcoRI i, were found to elevate TK expression in this assay. The stimulatory sequences were localised to to a 2. 6kb fragment, contained within the plasmid, pMCl. According to the mapping data of Hall et al. (1981) this region contained the complete sequences only of a 1.7kb transcript. The stimulatory effect was abolished by an 8bp linker insertion, which disrupted the reading frame of this gene, but not by other insertions within BamHI f. Induction mediated by the BamHI f fragment was confined to genes under the control of IE sequences, showing it to have the same specificity as the virion TIF, whereas EcoRI b mediated a general stimulatory effect. These experiments indicated that the 1.7kb gene within BamHI f encoded the TIF and that polypeptides encoded by EcoRI b were unlikely to be involved. Hybridisation of pMC6 (a subclone of pMCl) to infected cell RNA, prior to its translation in vitro, prevented the synthesis of a polypeptide of molecular weight 65,000. Immunoprecipitation of translated samples with monoclonal antibody identified this species as Vmw65, a major structural polypeptide. The gene encoding this polypeptide was found to possess an efficient promoter which could direct efficient expression in the absence of viral trans-activating polypeptides. The studies performed identify the virion factor responsible for trans-induction of IE genes as Vmw65, a major structural polypeptide, located in the tegument of the virion. Expression of HSV-1 regulated genes is not increased by infection with PRV, a related herpesvirus (Batterson and Roizman, 1983). Experiments were therefore carried out to examine the control of the PRV major IE gene. The 5' terminus of the raRNA was mapped by S1 nuclease analysis and hybrid plasmids, which contained IE upstream sequences linked to the HSV-1 TK gene, were constructed. Gene expression under the control of PRV IE or HSV-1 IE gene 3 upstream regions was compared using transient assays. It was found that infection with UV-irradiated PRV did not stimulate expression from PRV IE or from HSV-1 IE gene 3 upstream regions in BHK or pig kidney cells, indicating that PRV did not possess an effective TIF. Infection with UV-treated HSV-1, or cotransfection with pMCl (which encodes Vmw65) stimulated expression from both PRV and HSV IE gene upstream regions. However, co-infection with PRV and HSV-1 did not result in increased synthesis of the PRV IE polypeptide. The nucleotide sequence of the 5' end of the PRV transcript and its upstream region was determined. This region was unlike the upstream regions of HSV IE genes in overall structure, but showed a strong similarity to the enhancers of human and murine cytomegaloviruses (HCMV and MCMV). In particular, a reiterated 15bp element of the PRV upstream region was homologous to a conserved, repeated sequence element found in both HCMV and MCMV enhancer regions, and was also related to the "TAATGARATTC" motif found upstream of all HSV IE genes. In order to facilitate future investigations concerning the mechanism of action of the HSV-1 TIF, Vmw65, preliminary purification and characterisation were carried out. A polypeptide of molecular weight, 65,500 was partially purified from HSV-1 virions by treatment with NP40. Immunoprecipitation studies identified this species as the TIF, and also showed that a 65,000 molecular weight binding protein isolated from infected cells was unrelated. Vmw65 has no affinity for double-stranded calf thymus DNA, suggesting that it does not mediate transcriptional regulation by direct interaction with IE upstream sequences.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.795927  DOI: Not available
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