Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796378
Title: The control of varicella-zoster virus immediate early gene expression
Author: McKee, Thomas A.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1990
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Abstract:
Analysis of the cis-acting motifs and trans-acting factors responsible for regulating the expression of the varicella-zoster virus (VZV) major immediate early (IE) gene was undertaken. The control of VZV IE gene expression has relevance to the fields of eukaryotic gene regulation and herpesvirus biology. In order to analyse the control sequences of the VZV major IE gene, knowledge of the location of the 5' end of its mRNA was required. Primer extension and SI nuclease analyses concurred in defining it as being 74 base pairs (bp) upstream of the proposed translation initiation site. Analysis of DNA sequences upstream of the start site revealed a candidate for a 'TATA' motif present at -25 to -30 bp, a finding common to many genes transcribed by eukaryotic RNA polymerase II. Analysis of the activity of the control region of the VZV major IE gene was undertaken using plasmids in which sequences to -1150 were inserted upstream of the "reporter" genes encoding B-galactosidase or chloramphenicol acetyl transferase. The activities of the complete control region and of a series of deletions were then determined in a range of cell types, using short term transfection assays. A significant finding to emerge from these experiments was that the control sequences responsible for the transcription of the VZV major IE gene do not direct a high level of expression of reporter genes in any of the cell types tested. The baseline level of transcriptional activity was dependent on sequences located between nucleotides -131 and -25. Co-transfection of a plasmid which expresses the herpes simplex virus type 1 (HSV-1) protein Vmw65 resulted in a 20-50 fold stimulation of expression from the VZV IE promoter. This effect depended on the presence of regulatory sequences between -409 bp and -130 bp. A second set of co-transfection experiments investigated the effect on the VZV control sequences of a plasmid encoding the transactivating product of the adenovirus 5 ElA gene. The presence of this polypeptide resulted in stimulation of expression by 10 fold. In contrast with the effect of Vmw65, the activity of the ElA gene product depended on sequences present within the first 131 bp of the mRNA start site. Gel retardation and DNAase I protection assays were used to locate sites at which proteins bind to the control sequences. Results from these experiments reinforced and expanded the conclusions made from the functional assays. DNA fragments from the promoter region, -35 to -130, were shown to bind two proteins. Sequence analysis and competition experiments identified these proteins as belonging to two families of transcription factors known as 'CCAAT box binding' proteins and the 'ATF/CRE' family. A second protein binding region was present between -409 and -246 and was shown to consist of two binding sites for the cellular transcription factor Oct-1, which binds to the consensus ATGCAAAT. The proximal binding site could be expanded to incorporate a TAATGARAT motif known to allow Vmw65 to participate in the formation of a complex including Oct-1 and other cellular proteins, thus allowing Vmw65 mediated transactivation. Both sites were shown to bind Oct-1 and the proximal site was also shown to bind Vmw65 with high affinity, probably explaining the effect of this protein on VZV IE gene expression. In view of these results the analysis of VZV open reading frame (orf) 10 was undertaken. Sequence comparison of orf10 and Vmw65 had shown considerable homologies between the amino-terminal portions of the proteins but the absence from orf10 of a carboxy-terminal region of 78 amino acids known to be essential for transcriptional activation by Vmw65. Both the functional activity of orf10 and its ability to participate in complex formation at TAATGARAT motifs were tested. A plasmid, constructed to express the product of VZV orf10, failed to stimulate expression from plasmids containing HSV-1 or VZV IE control sequences. The product of VZV orf10 was also produced in vitro and failed to participate in the formation of DNA binding complexes when incubated with cellular proteins and the TAATGARAT motif. Two other VZV orfs were studied. The first was number 66, which encodes a protein which shows many similarities to known protein kinases. The system chosen to express this orf utilised the HSV-1 temperature sensitive mutant tsK and HSV-1 IE control sequences. At nonpermissive temperatures tsK overproduces IE polypeptides, generating quantities sufficient for biochemical characterisation of heterologous orfs expressed in this way. In order to simplify the selection of recombinant viruses containing VZV orfs the E. coli B-galactosidase enzyme, with HSV-1 IE control sequences, was inserted into the thymidine kinase gene of tsK.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796378  DOI: Not available
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