Expression and Characterization of Four Herpes Simplex Virus Type 1 Gene Products Required for the Replication of Viral DNA

Paterson, Janice Mackenzie (1991) Expression and Characterization of Four Herpes Simplex Virus Type 1 Gene Products Required for the Replication of Viral DNA. PhD thesis, University of Glasgow.

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Abstract

The work presented in this thesis is concerned with the study of the products of four genes of herpes simplex virus type-1 (HSV-1). These genes, UL5, UL8, UL9 and UL52, are members of a set of seven HSV-1 genes which are essential for the replication of HSV-1 DNA in tissue culture. Three of these seven genes encode products which are abundant in HSV-1 infected cells and have been relatively well characterised. These are UL29 which encodes the major single-stranded DNA binding protein, UL30 which encodes the viral DNA polymerase, and UL42 which encodes a double-stranded DNA binding protein, Mr 65 000. The protein products of the remaining four genes, UL5, UL8, UL9 and UL52, are of low abundance in HSV-1 infected cells and at the outset of this work had not been identified. Since then the UL9 gene product has been identified as an origin-binding protein which recognises specific DNA sequence elements (Olivo et al., 1988; Weir et al., 1989), and the UL5, UL8 and UL52 proteins have been purified as a complex from HSV-1 infected cells which has DNA helicase and DNA primase activities (Crute et al., 1989). To facilitate their identification and subsequent characterization, the UL5, UL8, UL9 and UL52 gene products were individually expressed in recombinant viruses. These four open reading frames (ORFs) were individually expressed under the control of the HSV-1 immediate-early (IE) 3 gene promoter in the temperature sensitive HSV-1 mutant tsK. A lesion within the IE3 gene of this mutant results in the overproduction of IE gene products at the non-permissive temperature (NPT). Thus, products of the inserted replication genes could be expressed in the absence of other DNA replication proteins at the NPT. Each ORF was also expressed under the control of the polyhedrin promoter in recombinant baculoviruses constructed by Dr N D Stow. Insertion of these ORFs into tsK recombinant viruses allowed the products of the UL5, UL8, UL9 and UL52 genes to be identified as novel polypeptides on SDS-polyacrylamide gels. The apparent molecular weights of the over-expressed proteins corresponded closely to those predicted from DNA sequence analysis. The gene products were also identified immunologically by reactivity with antisera raised against peptides corresponding to the C-termini of their predicted amino acid sequences (a kind gift of Dr M D Challberg). On the basis of [32P]-orthophosphate labelling experiments, none of the four proteins appeared to be significantly phosphorylated, . Based upon the identification of a UL5, UL8 and UL52 protein complex in HSV-1 infected cells (Crute et al. , 1989), attempts were made to assemble this complex in vivo, initially by infecting BHK cells with the tsK recombinant viruses, and subsequently by infecting insect cells with recombinant baculoviruses. The enzymatic activities of the complex were investigated using the baculovirus system. Insect cells were infected with either parental baculovirus or the UL5, UL8 and UL52 recombinants, individually or in all possible combinations. Extracts from the infected cells were fractionated by phosphocellulose column chromatography assayed for DNA-dependent ATPase, DNA helicase and DNA primase activities. Novel peaks of each of these activities were detected in extracts from cells triply infected with the UL5, UL8 and UL52 recombinants or doubly infected with UL5 and UL52 recombinants. These activities, which were not present in any of the other extracts, eluted at 150-200mM NaCl, similar to that reported for the HSV-1 helicase-primase detected in cells infected with wt HSV-1 (Crute et al. , 1988). SDS-PAGE analysis of the phosphocellulose column fractions revealed that the UL5, UL8 and UL52 proteins co-eluted with the induced activities, at salt concentrations differing from those at which any of these proteins eluted when expressed alone. This result is indicative of an interaction between the three proteins. The UL5 and UL52 proteins co-eluted in a similar fashion when expressed in the absence of the UL8 protein, at a position co-incident with the novel peaks of enzymatic activity. The UL8 protein is therefore not required for the novel DNA-dependent ATPase, DNA helicase and DNA primase activities associated with the complex, and moreover, the UL5 and UL52 proteins together are able to form a functional complex in the absence of the UL8 protein. The intracellular localization of the HSV-1 origin-binding protein (OBP), which is encoded by gene UL9, and the HSV-1 helicase-primase complex were studied by indirect immunofluorescence in BHK cells infected with the tsK recombinant viruses. OBP expressed in the absence of other DNA replication proteins localized within the nucleus where it exhibited a diffuse pattern with discrete foci also present. Localization of the helicase-primase complex was investigated by fluorescently staining the UL52 protein subunit. When expressed alone, the UL52 protein exhibited a cytoplasmic, perinuclear staining pattern. This pattern remained unchanged when the UL52 protein was coexpressed with either the UL5 or UL8 protein. However, when the UL5, UL8 and UL52 proteins were expressed together the UL52 protein was efficiently localized within the nucleus. These results suggest that the UL8 protein, which is not required for the enzymatic activities of the complex, may have a role in facilitating entry of an active complex into the cell nucleus.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78348
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:32
Last Modified: 30 Jan 2020 15:32
URI: http://theses.gla.ac.uk/id/eprint/78348

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