Characterisation of the HSV-1 DNA Packaging Protein Encoded by the UL25 Gene

Targett-Adams, Paul (2001) Characterisation of the HSV-1 DNA Packaging Protein Encoded by the UL25 Gene. PhD thesis, University of Glasgow.

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Herpes simplex virus type 1 (HSV-1) DNA replication results in the formation of head- to-tail concatemers which are cleaved into genome size units and packaged into the procapsid in the nuclei of virus-infected cells. The procapsid is a spherical structure with icosahedral symmetry and contains an internal protein scaffold which is removed at the same time viral DNA is encapsidated. During the DNA packaging process the procapsid angularises and the DNA-containing capsid can subsequently mature into an infectious virion. The product of the HSV-1 UL25 gene is a minor component of the viral capsid and has been implicated in the HSV-1 DNA packaging process. The overall goal of this thesis was to investigate the role of the UL25 protein in the HSV-1 lytic cycle. Before a detailed study of this protein could be undertaken, a number of reagents had to be prepared, including potent UL25-specitlc antibodies. Therefore, an initial aim of the project was to express the UL25 protein in a variety of in vivo recombinant protein expression systems and to purify the soluble recombinant UL25 protein for use as an antigen in the production of UL25-specific monoclonal antibodies. Maltose binding protein (MBP)-tagged UL25 and polyhistidine (His)-tagged UL25 were expressed in Escherichia coli and recombinant baculovirus-infected Sf21 cells respectively. BALB/c mice were immunised with purified soluble MBP-UL25 fusion protein and given a final boost with purified soluble His-tagged UL25 protein. Twelve hybridoma cell lines secreting UL25-specific monoclonal antibodies were isolated. The monoclonal antibodies were characterised using Western blot, immunoprecipitation and immunofluorescence assays. From this analysis a monoclonal antibody that reacted strongly with the UL25 protein in each of the immunoassays was purified for use in subsequent experiments. In the absence of other HSV-1 proteins, UL25 localised predominantly to the cytoplasm of cells transiently expressing the protein. In cells infected with HSV-I, however, UL25 protein was concentrated in the nuclei at late times. To investigate whether the HSV-1 capsid shell proteins, VP5, VP23 and VP19C, were required for the nuclear localisation of UL25 in HSV-I-infected cells, the distribution of UL25 protein was examined in cells infected with HSV-1 mutants which fail to express these proteins. In non-complementing cells infected with VP23 or VPS null mutants, the distribution of UL25 protein was similar to the pattern in wild-type (wt) virus-infected cells indicating that neither VP23 nor VP5 were necessary for the nuclear localisation of the UL25 protein during HSV-1 infection. Since capsid assembly did not occur under these conditions (Desai et al., 1993), nuclear localisation of UL25 was independent of capsid assembly. The intracellular distribution of UL25 was also examined in non-complementing cells infected with a VP19C null mutant of HSV-1. However, this virus appeared to have an additional mutation, one which affected late viral protein production, and no conclusive results were obtained through the use of this virus. The localisation of the UL25 protein was therefore investigated in cells infected with ts2, a mutant of HSV-1 that contains a temperature sensitive (ts) lesion in the VP19C protein. In cells infected with ts2 at the non-permissive temperature (NPT), UL25 co-localised with the capsid shell proteins at the perinuclear region of cells with little, if any, UL25 protein observed in the nuclei. These findings suggested that the VP19C protein was necessary for the nuclear distribution of UL25 during wt HSV-1 infection. However, in cells infected with a ts2 marker rescuant at the NPT, UL25 remained localised to the perinuclear region while the capsid shell proteins were found in the nuclei. This result indicated that the altered intracellular distribution of UL25 in cells infected with ts2 at the NPT was not a consequence of the ts lesion in the VP19C protein. Furthermore, UL25 also localised to the perinuclear region of cells infected with HSV-1 A44, the parental syncytial strain of ts2, at the NPT. This virus formed syncytia to a greater extent in cells infected at the NPT compared to the permissive temperature and it is possible that the altered intracellular distribution of UL25 protein in cells infected with HSV-1 A44 at the NPT may have resulted from the formation of syncytia or from an aberrant interaction with a component of the HSV-1 tegument. The association of UL25 protein with the capsid was initially examined using the recombinant baculovirus expression system to obtain information about the copy number and the location of the UL25 protein in the capsid as well as its interaction with capsid shell proteins. The UL25 protein was incorporated into capsids generated in insect cells multiply infected with recombinant baculoviruses expressing the HSV-1 capsid shell, scaffolding and the UL25 proteins, suggesting that the UL25 protein can interact with capsids in the absence of other viral proteins. This finding is in agreement with earlier results of McNab et al. (1998). (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Valerie Preston
Keywords: Virology, Genetics
Date of Award: 2001
Depositing User: Enlighten Team
Unique ID: glathesis:2001-76258
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:13
Last Modified: 19 Nov 2019 16:13

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