Studies on the US3 and UL13 Protein Kinase Genes of Herpes Simplex Virus Type 1

Coulter, Lesley Jayne (1993) Studies on the US3 and UL13 Protein Kinase Genes of Herpes Simplex Virus Type 1. PhD thesis, University of Glasgow.

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The availability of the complete sequence of HSV-1 has permitted interpretation of HSV-1 gene function. As a result of database searches, US3 and UL13, the two HSV-1 genes studied during the course of this research, were found to possess amino acid sequence motifs characteristic of the protein kinase family. The US3 gene has indeed been shown to encode a protein kinase present in the cytoplasm of infected cells, however, protein kinase activity has not yet been assigned to the UL13 gene product, although the protein does correlate with a novel protein kinase activity in the nuclei of HSV-1 infected cells. Protein kinases constitute a very important class of enzymes which are responsible for the regulation of many cellular processes, thus, it is considered likely that these two genes play a significant role in the life cycle of HSV-1. This was investigated by constructing HSV-1 viruses which contain the US3 or UL13 genes disrupted by the insertion of the Escherichia coli lacZ gene. This insertional mutagenesis technique has been used successfully to investigate the functions of several other HSV-1 genes. During the course of this research three lacZ insertion mutants were constructed, a US3-lacZ insertion mutant, a UL13-lacZ insertion mutant and a UL13-US3 double insertion mutant. The effects of these mutations on the growth properties of HSV-1 and on the phosphoprotein profiles of infected cell extracts and virion preparations were then investigated. The US3-lacZ insertion mutant used in the experiments described in this thesis was found, at a late stage of the work, to be contaminated with a low level of wt virus, despite 5/6 rounds of plaque purification. Nevertheless, the titres of the US3-lacZ virus were significantly reduced compared to wt, indicating that, although the gene is not essential for virus growth, its disruption does impair virus growth. This reduction in growth does not appear to be due to a decrease in the amount of DNA synthesised by the US3-lacZ virus but to a decrease in the infectivity of the virus particles (as indicated by the significantly higher particle:pfu ratio for the US3-lacZ virus). Examination of the phosphoprotein profiles of US3-lacZ infected cells, following in vitro phosphorylation, showed a reduction in the degree of phosphorylation of several proteins with estimated MWs of 200K, 80K and 30K. These proteins, which represent potential substrates of the US3 protein kinase, have not yet been identified, although the 30K protein may correspond to the UL34 gene product, which has recently been reported to be a substrate of the US3 protein kinase. The growth of the UL13-lacZ insertion mutant, both in vitro and in vivo, does not differ significantly from that of wt, indicating that UL13 is not essential for virus growth, despite its conservation throughout the three herpesvirus families. Comparison of silver-stained gels of wt and UL13-lacZ virion preparations identified the 57K UL13 gene product as a relatively abundant component of the tegument, probably corresponding to VP18.8 - a phosphoprotein previously mapped to this region of the genome. The phosphoprotein profiles of both ULl3-iacZ-infected cells (nuclear extracts) and UL13-lacZ virions showed a reduction in the phosphorylation of several proteins, of MW 160K, 106K, 60K, 45K and 38K, indicating that the UL13 gene product does possess protein kinase activity. One of these potential substrates of UL13, the 38K phosphoprotein, has been mapped, using intertypic recombinants, to gene UL49, the product of which is known to be the virion tegument protein VP22. UL13-lacZ infected cell nuclear extracts were also found to contain two hyper- phosphorylated proteins with estimated MWs of 45K and 70K. The origins of these proteins are not known. Preliminary experiments with the UL13-US3 double insertion mutant have indicated that the growth of the virus is impaired to a much greater extent than expected from the growth properties of the two single lacZ mutants. One possible explanation for this is that the two kinases have common substrates and can therefore substitute for each others absence in the two single mutants, however this is considered unlikely as the phosphoprotein profiles of the two single mutants have not revealed any common substrates. Alternatively, the possibility exists that the two kinases may carry out similar functions in the virus life cycle. Thus, the research presented in this thesis provides evidence that the UL13 gene product, a relatively abundant virion protein, possesses protein kinase activity, and identifies the UL49 gene product as a major substrate for this activity. The research has also indicated several potential substrates for the US3 protein kinase, although these have not yet been identified. Finally, preliminary experiments with the UL13-US3 double insertion mutant have revealed a considerable reduction in virus growth compared to the two single mutants.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John H Subak-Sharpe
Keywords: Virology
Date of Award: 1993
Depositing User: Enlighten Team
Unique ID: glathesis:1993-75331
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:00
Last Modified: 19 Nov 2019 21:00

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