The Encapsidation of Herpes Simplex Virus Type 1 DNA

Hodge, Paul Daniel (1999) The Encapsidation of Herpes Simplex Virus Type 1 DNA. PhD thesis, University of Glasgow.

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The herpes simplex virus genome contains an approximately 400 bp direct repeat at its ends known as the 'a' sequence, which contains all of the signals necessary for the cleavage and packaging of concatemeric replication products. During replication the genome is circularised, probably by direct ligation of the termini, generating a novel junction between two tandemly repeated 'a' sequences. We have cloned such a novel junction and in agreement with previous results (Nasseri & Mocarski, 1988), have shown that it can serve as a substrate for cleavage and packaging. The novel junction contains two highly conserved regions of the 'a' sequence which have been shown to be essential components of the packaging signal (pad and pac2). This thesis describes the introduction of specific mutations in this region and the development of a transient packaging assay to examine the effect of these mutations. In addition the mutants were tested for their ability to be serially propagated, extending the analysis beyond cleavage and packaging. Mutations were made using the motifs previously described by Deiss et al (1986). The pad and pac2 regions as a whole were deleted using convenient restriction enzyme sites and individual motif mutations, were introduced using a site directed mutagenesis method based on that described by Kunkel et al. (1991). The mutant sequences were then tested for their ability to direct cleavage and packaging in the transient packaging assay. This identified that both the pad and pac2 sequences as a whole represent essential components of the packaging signal. Both the location and the sequence of the pac2 T rich element were also shown to be essential in a functional cleavage and packaging signal. In addition a substitution mutation of the pac2 unconserved region reduced the efficiency of packaging directed by wt HSV-1 strain 17 but no effect on packaging efficiency was observed when HSV-2 strain HG52 was used. This result appears to indicate an unexpected divergence between HSV-1 and HSV-2. No function was attributable to either the conserved pac2 consensus sequence or any of the individual motifs of the pad sequence. However, considering the importance of the pad region as a whole the lack of identifiably important motifs is likely to reflect a level of redundancy within the pad region as opposed to a lack of biological function. Finally the mutants were used to examine the signals necessary for the serial propagation of defective genomes. This revealed that both GC rich regions of pad are involved in the serial propagation of defective genomes. No sequences within pac2 were identified as important. However, this may reflect either redundancy within the region or the importance of sequences which are also involved in DNA packaging.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Duncan McGeoch
Keywords: Virology
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-76344
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 15:23
Last Modified: 19 Nov 2019 15:23

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