Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796271
Title: Studies of the protein kinases of α-herpes viruses
Author: Stevens, Rowena
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1989
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Abstract:
This work concerns the two similar protein kinase activities---HSV-PK and PRV-PK---which are found in cultured animal cells infected with herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV), respectively; and two genes, predicted from their sequence to encode protein kinases: US3 in HSV-1 and pk in PRV. The aims of this work were to determine the relationship between pk and PRV-PK, and to investigate the location and possible substrates of the protein kinases. First, anti-sera were raised against the protein products of the US3 and pk genes in the form of fusion-proteins with B-galactosidase in E. coll. A 0.8kb Sal I fragment of the US3 gene and a 0.8kb Pst I fragment of the pk gene were separately cloned into appropriate sites in the B-galactosidase gene of the plasmid vector pUEX-2, and E. coli cells were transformed with the resulting recombinants. The fusion-proteins were induced, partially purified by gel exclusion chromatography, injected into rabbits and anti-sera were prepared. PRV-PK was purified to homogeneity by successive chromatography on DEAE-cellulose, TSK phenyl-5PW, threonine-Sepharose and protamine- agarose. During the purification, protein kinase activity coeluted with material of 38kDa, found only in infected cells, which showed immunoreactivity towards the anti-serum raised against the protein product of the pk gene of PRV. This demonstrated that PRV-PK is encoded by the pk gene of PRV, and is consistent with previous work which had indicated that the US3 gene of HSV-1 encodes HSV-PK. This anti-serum also cross-reacted with a cellular protein of 34kDa which was resolved from PRV-PK during chromatography on DEAE-cellulose. As a result of this cross-reactivity, the anti-serum was unsuitable for immunochemical studies which might have determined the location of the enzyme within intact infected cells. The anti-sera were used to investigate the location of PRV-PK and HSV-PK within their respective virions. Purified virions were subjected to polyacrylamide gel electrophoresis and the proteins transferred to nitrocellulose. The anti-serum raised against the product of the US3 gene reacted with two proteins of 68kDa and 69kDa, as was also observed for extracts from infected cells. This indicated that HSV-PK is present within the virions of HSV. The anti-serum raised against the product of the gene, pk, reacted with a single protein of 38kDa, demonstrating that PRV-PK is present within the virions of PRV. This anti-serum also reacted with purified nucleocapsids and with virions which had been treated with the non-ionic detergent, NP-40, to remove the envelope of the virion. This implies that PRV-PK is associated with the nucleocapsid, which may be important in determining the physiological substrate(s) of the viral kinase. Possible viral substrates of PRV-PK within PRV virions were investigated. Purified virions were incubated with gamma[32P] ATP, subjected to gel electrophoresis and the phosphorylated proteins detected by autoradiography. The major phosphorylated protein had a molecular weight of 112000. This protein was also phosphorylated when virions, in which the endogenous protein kinases had been inactivated by heat, were incubated with exogenous PRV-PK in the presence of gamma[32P]ATP, demonstrating that the 112kDa protein can be a substrate for PRV-PK in vitro. When cells were incubated with [32P]orthophosphate during infection in vivo, the 112kDa protein was also phosphorylated. This suggested that the 112kDa protein may be a physiological substrate of PRV-PK. However, at least three other distinct protein kinases have been identified in PRV virions, one of which, casein kinase II, can also phosphorylate this virion protein in vitro. When PRV virions were incubated with y[32P]ATP in the presence of 0.5M KCl, which inhibits casein kinase II but not PRV-PK, the phosphorylation of the 112kDa protein was inhibited. This implies that casein kinase II is responsible for most of the phosphorylation of the 112kDa protein in isolated virions in vitro, but does not exclude the possibility that the protein is a substrate for PRV-PK in vivo. However, immunochemical assays showed that PRV-PK is at least 100 times more abundant in the cytosol of infected cells than within virions, so that it seems more likely that the physiological role of PRV-PK involves the phosphorylation of proteins within the cytosol of infected cells. An attempt was made to detect possible cellular substrates for HSV-PK by expressing the US3 gene constitutively in eukaryotic cells, after introducing it into the cellular genome. A 2.8kb fragment of DNA which included the promoter and coding region for US3 was cloned into the vector, Homer 5. The recombinants were transformed into 208F rat fibroblasts by calcium phosphate precipitation, and transformants selected by resistance to geneticin, which was conferred by the vector. Extracts from the transformed cell lines were subjected to chromatography on DEAE-cellulose and assayed for HSV-PK activity, but this could not be detected. Cell lines expressing greater quantities of HSV-PK will be required before this approach to identify a possible cellular substrate can be executed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796271  DOI: Not available
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