Phosphorylation of the Herpes Simplex Virus Type 1 UL13 Protein Kinase and a Putative Target Protein Encoded by Gene UL49

Antrobus, Philip Robin (2000) Phosphorylation of the Herpes Simplex Virus Type 1 UL13 Protein Kinase and a Putative Target Protein Encoded by Gene UL49. PhD thesis, University of Glasgow.

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Protein kinases (PKs) are ubiquitous enzymes which utilise phosphorylation, a reversible post-translational modification, to regulate protein activity. Protein phosphorylation is widespread, affecting nearly all aspects of growth and homeostasis in eukaryotic cells. However, over twenty years ago certain viral transforming genes were found to possess PK activity, bringing protein phosphorylation to the attention of virologists. Sequence analysis of the herpes simplex virus type 1 (HSV-1) genome identified two genes, US3 and UL13, whose products exhibit PK-specific amino acid sequence motifs. The US3 PK has been purified, the enzyme activity characterised and a number of targets for phosphorylation identified. The UL13 PK has not yet been purified, but its enzymatic characteristics and potential target proteins have been investigated. It localises in the nuclei of infected cells, utilises ATP or GTP to phosphorylate acidic but not basic substrates, and is a minor component of the virion tegument. The UL13 PK activity is stimulated by high salt concentration and is insensitive to inhibition by heparin. Several proteins have been identified as targets for the UL13 PK, one being the abundant viral tegument protein encoded by HSV-1 gene UL49. The aims of this project were to confirm that the UL13 PK targets the UL49 protein for phosphorylation, to characterise further the UL13 PK activity, and to map phosphorylated residues within the UL13 and UL49 proteins. From an analysis of three independent UL13 mutants, each with a lesion in a different region of the UL13 gene, it was confirmed using an in vitro assay that phosphorylation of the UL49 protein was dramatically reduced in the absence of a functional UL13 protein. In radiolabelled nuclear extracts of wt HSV-1 infected cells or membrane-stripped wt virions, the UL13 protein was detected as a highly radiolabelled 57 kDa phosphoprotein. Phosphorylation of this protein was stimulated by increasing salt concentrations. Thus, HSV-1 UL13 PK differs significantly from the previously characterised HSV-2 UL13 PK in both its salt sensitivity and optimum pH. Radiolabelling of the UL13 PK maybe attributed to autophosphorylation. However, at least two hyperphosphorylated forms of the UL13 protein were detected in radiolabelled nuclear extracts incubated with excess unlabelled ATP or GTP. The level of hyperphosphorylation differed between cell lines, and hyperphosphorylated UL13 protein was not detected in radiolabelled membrane-stripped virions. Hyperphosphorylation was insensitive to heparin, but was completely abolished in the presence of a potent inhibitor of cellular casein kinase II. The most obvious target for the UL13 PK identified during this study was a 38 kDa virion protein, which was confirmed by mass spectrometry and by use of a mutant virus expressing a C-terminally truncated form of the UL49 protein to be encoded by HSV-1 gene UL49. The UL49 protein was still phosphorylated, albeit to a greatly reduced level, in nuclear extracts prepared from UL13 mutant-infected cells. The additional PK or PKs targeting the UL49 were inhibited by heparin and sensitive to increasing salt concentrations. This suggests that a cellular PK targets the UL49 protein. However, an inhibitor of casein kinase II had little effect on phosphorylation of the UL49 protein. Initially, the UL49 protein was found to cycle phosphate, and did not appear to be hyperphosphorylated beyond a basal level. However, under altered buffer conditions, it was hyperphosphorylated and also possibly nucleotidylylated, these modifications seeming to require sequences near the C-terminus of the UL49 protein. Phosphopeptide mapping experiments performed on the UL49 protein phosphorylated at low salt concentrations led to detection of a 2 kDa tryptic phosphopeptide by SDS- PAGE, and mass spectrometric analysis identified a phosphorylated 1703.91 Da tryptic peptide which was phosphorylated at high salt concentrations. It is likely that these two peptides represent cellular and viral PK target sites. Mapping of the UL13 protein suggested that phosphopeptide fragments are located in the serine-rich, N-terminal 100 residues of the protein, a region which lies outside the established catalytic domain of the UL13PK. As well as providing data in support of UL13 encoding a PK which targets the UL49 protein for phosphorylation, the data presented in this thesis represents a further characterisation of the UL13 PK activity and provides new insights into phosphorylation of the UL49 and UL13 proteins.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Andrew Davison
Keywords: Virology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-76012
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15

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