Studies on herpes simplex virus type 1 DNA packaging
Herpes simple text 1 (HSV-1)has a linear double- stranded DNA genome of approximately 152 kpb. The products of DNA replication are molecular weight concatemers consisting of tandem head-to-tail repeats of the viral DNA. The work described in the thesis relates to the mechanisms by which this concate...
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| Format: | Dissertation |
| Language: | English |
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ProQuest Dissertations & Theses
01-01-2005
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| Online Access: | Get full text |
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| Summary: | Herpes simple text 1 (HSV-1)has a linear double- stranded DNA genome of approximately 152 kpb. The products of DNA replication are molecular weight concatemers consisting of tandem head-to-tail repeats of the viral DNA. The work described in the thesis relates to the mechanisms by which this concatemeric DNA is cleaved into unit length genomes and inserted into a performed procapsid structure which eventually matures into the infections virion. A series of experiments were performed with the HSV-1 temperature sensitive mutant ts1201 which carries a mutation in the viral maturational protease (Preston et.al,1983) .In cells infected at the non-permissive temperature this mutant replicates but fails package its genome. However, upon downshift to the permissive temperature the defect is reversible and DNA packaging and production of infectious virus can occur. I confirmed these findings using Southern blot hybridisation, and demonstrated that neither viral protein nor DNA synthesis after down shift were required for DNA packaging. It was concluded that packaging of the HSV-1 genome takes 30-60 minutes, and result were consistent with a model in which the direction of packaging was from the long (L) to short (S) terminus. An experiment in which the cells were back to the non-permissive temperature suggests that the activity of the protease was not continuously required for DNA packaging. Transistent assays based on amplicons (bacterial plasmids containing a functional HSV-1 orgin of replication and packaging signals) have been usefyul in analysing the viral sequences required for packaging (Hodge & Stow, 2001, Stow et al, 1983). Transfection of the amplicon being replicated and packaged into virus particles. This assay previously localised the packaging signals within the viral a sequence at either end of the genome (Stow et al., 1983), and showed that the minimum packaging signal corresponded to an approximately 200 bp fragment representing the novel junction between two tandem a sequences (the Uc-DRl-Ub fragment) (Hodge & Stow, 2001). I used two mutagenesis approaches to analyse the importance of sequences within the Uc-DRl-Ub fragment for DNA packaging. Mutants containing deletions extending various distances from the Ub end were created using nuclease Bal31 or the restriction enzyme Dral, and tested in the amplicon assay. The results indicate that the DRl element is probably important for fully efficient packaging in the context of the minimum packaging signal, despite previous evidence that its sequence was dispensable (Varmuza & Smiley, 1985, Smiley et al., 1992). In addition there appears to be a functional redundancy amongst the pad motifs located within Ub. A transposon mutagenesis procedure was used to generate a panel of amplicons containing insertions throughout the Uc-DRl-Ub region which were also tested in the amplicon assay. This allowed the identification of regions in which insertions either greatly inhibited, or had relatively little effect upon, DNA packaging.Although the amplicon assay is convenient, it would be advantageous to be able to analyse packaging signals in the context of the viral genome. To develop such a system I used a copy of the HSV-1 genome cloned as a bacterial artificial chromosome (BAC) from which the packaging signals had been deleted. This BAC (HSV-1 BAC21) contains a unique Pacl restriction enzyme site into which presumptive packaging signals can be cloned. I generated a transfer vector (pATLG) into which packaging signals from my amplicons could be cloned and subsequently excised with Pacl for cloning into HSV-1 BAC21. Optimal conditions for cloning the small packaging signals into the large BAC were identified. Using this approach I cloned the wild-type Uc-DRl-Ub fragment into HSV-1 BAC21 in both orientations, generating BAC LG 19 and BAC LG20. When permissive cells were transfected with HSV-1 BAC21 no infectious virus was produced. In contrast transfection of BAC LG 19 and BAC LG20 gave rise to virus plaques, and stocks of the recovered virus were prepared and analysed. These results demonstrated that the minimum packaging signal, Uc-DRl-Ub is functional in the context of the viral genome when inserted in either orientation. However the yield of virus was significantly reduced compared to controls containing the a sequence in its usual locations. Nevertheless, this approach should provide a convenient way for studying mutations within the packaging signal in the context of the full length HSV-1 genome. |
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| ISBN: | 035568554X 9780355685541 |