Analysis of 5 Genes Unique to Equine Herpesvirus 1 and Characterisation of the Products of Genes 67 and 71

Sun, Yi (1996) Analysis of 5 Genes Unique to Equine Herpesvirus 1 and Characterisation of the Products of Genes 67 and 71. PhD thesis, University of Glasgow.

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Equine herpesvirus 1 (EHVl), a member of the alphaherpesvirinae, is a significant viral pathogen of horses causing a variety of clinical problems including respiratory disease, abortion and neurological disorders. The complete DNA sequence of EHVl strain Ab4, a pathogenic UK isolate has been determined. There are 81 open reading frames (ORF) predicted from the DNA sequence. Most of these predicted gene products have not been identified and their functions in the virus life cycle are not known. Among these putative genes, five 1, 2, 67, 71 and 75 had no detectable homologues in HSVl and VZV,and appeared to be unique to EHVl (Telford et al., 1992). This feature suggested that these genes may have specific roles in the virus life cycle in the horse. The research presented in this thesis has concentrated on investigating the role of the five genes in the virus life cycle. The project included three parts: (1) Construction and characterisation of deletion mutants in the target genes. For construction of deletion mutants the majority of the encoding sequence of each target gene was removed and replaced by the E. coli lacZ gene as a marker gene. The isolation of viable mutants in the five target genes demonstrated that none of these genes were essential in the virus life cycle in vitro. Their growth characteristics, host range and temperature sensitivity in tissue culture were indistinguishable from those of wild type virus with the exception of the gene 71 deletion mutant (ED71). ED71 grows less well than wild- type virus and its revertant, produces smaller plaques and has an obviously higher particle/pfu ratio of progeny virus than the wild-type virus and revertant in vitro. (2) Identification and characterisation of the target gene products. To raise specific antisera the genes were separately expressed as 6-galactosidase fusion proteins in E.coli. These consisted of amino acids 4-202 of the putative gene 1 protein, amino acid sequence 2-205 of the putative gene 2 protein, amino acids 12-260 of the putative gene 67 protein, amino acids 434-797 of the putative gene 71 protein and amino acids 14-130 of the putative gene 75 protein. Two antisera, anti-67 and anti-71, have been raised against the gene 67 and gene 71 fusion proteins in rabbits. Use of the deletion mutants in conjunction with the antisera. allowed the unambiguous identification of the protein products of genes 67 and 71. Anti-67 serum specifically recognised a 36KD Mr polypeptide in infected cell extracts which was absent from mock and mutant ED67 infected cell extracts and was not recognised by the pre- immune serum. In the lytic replication cycle, the protein is regulated as an early polypeptide and modified as a phosphoprotein. With immunofluorescence and cellular fractionation experiments, the protein has been shown to be associated with intracellular membranes and distributes as novel ribbon or filament like structures, concentrated in a perinuclear location within the cytoplasm of infected cells. The protein does not co-localise with either tubulin, actin or Golgi bodies. However, the precise localisation of the gene 67 protein within cells is unclear. The gene 67 protein is also a component of virions and is located in the nucleocapsid/tegument. Anti-71 serum, specifically recognises a 192KD Mr infected-cell polypeptide. This protein is absent from mock and mutant ED71 infected cell extracts and is not recognised by pre-immune serum. This result confirmed that the 192KD polypeptide was encoded by EHV1 gene 71. Gene 7 lis regulated as a leaky late gene during EHV1 lytic infection. The 192 KD gene 71 protein is associated with cellular membranes and is also a component of the virion envelope with a Mr > 200KD. Post-translational modification experiments showed that the gene 71 protein was sensitive to tunicamycin and monensin; the Mr of the protein was greatly reduced by deglycosylation with exoglycanases, which demonstrated that the gene 71 protein was post-translationally modified as a glycoprotein with limited N and heavy O-linked glycosylation. These features of the gene 71 protein indicate that the protein is a class I membrane glycoprotein which is consistent with the features of the gene 71 protein predicted from sequence analysis (Telford et al., 1992a). During the course of this study, it became clear that the gene 71 protein had very similar properties to that of gp300, a EHV 1 glycoprotein, which had been characterised as being heavily O-linked with a Mr over 400 KD in SDS-PAGE linked by DATD. gp300 had been previously found to be encoded by EHVl gene 28 (Whittaker et al., 1990; 1992a). To determine the relationship between the gene 71 protein and gp300, a comparative experiment was carried out with anti-71 and a monoclonal antibody, P19, which specifically recognised gp300 (Whittaker, et al., 1992a). The comparative data shows that gp300 and the gene 71 protein have the same molecular weights under the same gel conditions and are both absent from ED71 infected cell extracts, but restored in ED71 revertant infected cell extracts. This data clearly confirmed that gp300 was encoded by EHV1 gene 71. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: M S Brown
Keywords: Virology, Genetics
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-76106
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:39
Last Modified: 19 Nov 2019 16:39

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