Herpes Simplex Virus DNA-Binding Proteins: Studies on 21K and the 'a' Sequence

MacLean, Christine Anne (1987) Herpes Simplex Virus DNA-Binding Proteins: Studies on 21K and the 'a' Sequence. PhD thesis, University of Glasgow.

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This project has involved the investigation of the polypeptide products of gene US11, and their involvement in DNA-protein interactions occurring within the 'a' sequence of HSV-1. The work stems from the observation of Dalziel and Marsden (1984), that polypeptides of apparent molecular weight 21,000 and 22,000 (21K, 22K) specifically interacted with the HSV-1 'a' sequence - a sequence involved in a number of functions, including circularisation of the virus genome, inversion between the L and S segments of the genome, and cleavage and packaging of viral DNA. To characterise the 21K/22K polypeptides, antisera were raised against synthetic oligopeptides corresponding to five different regions of the predicted US11 gene product, since earlier evidence suggested that the 21K and 22K polypeptides were the products of this gene. All five oligopeptides were immunogenic, and four induced antisera which appeared to recognise virus-specific polypeptides. The polypeptides recognised included species with apparent molecular weights of 22K, 21K, 17.5K, 15K, 14K and 11K, and these were shown, by tryptic peptide analysis, to share common amino acid sequences. Timecourse experiments, and the use of phosphonoacetic acid, an inhibitor of viral DNA replication, established that the 21K and 22K polypeptides are true late gene products, and immunoblotting of proteins eluted from DNA cellulose demonstrated that they are also DNA-binding proteins. Immune electron microscopy studies, however, demonstrated that the US11 gene products localise strongly to nucleoli of infected cells - an unexpected location for an 'a' sequence binding protein, since viral DNA is absent from nucleoli (Rixon et al. , 1983). Attempts to purify the 21K polypeptide, with a view to studying in more detail its interaction with the 'a' sequence, met with little success. Therefore, DNase I footprinting experiments on the HSV-1 'a' sequence were carried out, using crude whole cell extracts. The experiments revealed DNA-protein interactions occurring within two regions of the 'a' sequence - the DR2 repeat elements and the region. These interactions were specific for virus-infected cell extracts. Within the DR2 repeat region a repetitive pattern was seen, consisting of an area of protection (spanning seven nucleotides - GGGGAGG), flanked on either side by two nucleotides (AG and GG) at which cleavage was enhanced, and separated by one unaffected nucleotide (C). Within the region there was no apparent protection, however, there was a very marked increase in cleavage between a number of nucleotides. The crude whole cell extracts possessed both a 5'--3' exonuclease and a DNase I-like activity which limited the usefulness of the footprinting assay : it was therefore not possible to determine whether specific cleavage events were generating the enhanced cleavages observed. Use of an HSV-2 deletion mutant lacking gene Us11 demonstrated that the products of this gene are not involved in these interactions. Timecourse and drug inhibition experiments suggested that early polypeptides are involved, while experiments involving a temperature sensitive mutant tsK showed that immediate-early polypeptides, with the possible exception of Vmw175, are not sufficient alone to produce the observed interactions. The HSV-1 and HSV-2 'a' sequences are heterogeneous and share only limited stretches of homology (Davison and Wilkie, 1981). The most extensive region of homology lies within Ub, and overlaps the region of enhancement. The use of a cloned synthetic oligonucleotide representing this homology region, and an intact HSV-2 'a' sequence, suggested that the homology region is involved in the interactions observed . Enhancement was only observed on 3' end-labelled DNA fragments. For technical reasons, it was only possible to 3' end-label one strand of the intact HSV-1 'a' sequence close to the region, and one strand (the opposite strand) of the cloned synthetic oligonucleotide representing the homology region. Comparison of the patterns of enhancement observed on these two substrates led to the conclusion that the enhanced cleavages could reflect the 5' boundaries of protein binding sites, revealed by an endogenous 5'-3' exonuclease activity. Two putative Ub-binding sites are proposed, one contained entirely within, and the other overlapping, the homology region. These have the sequence 5' -CCAAAACCCCCCC-3.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1987
Depositing User: Enlighten Team
Unique ID: glathesis:1987-77566
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53
URI: https://theses.gla.ac.uk/id/eprint/77566

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