Characterisation of Herpes Simplex Virus Type 1 ts Mutants Which Have Structural Defects

Addison, Caroline (1986) Characterisation of Herpes Simplex Virus Type 1 ts Mutants Which Have Structural Defects. PhD thesis, University of Glasgow.

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Abstract

Four temperature-sensitive DNA+ mutants, ts1201, ts1203, ts1204 and ts1208, each of which contain a lesion in HSV DNA fragment EcoRI f (mu 0.312-0.415), have been characterised in this study. Ts1204 has a temperature-sensitive lesion located within a 400bp region between mu 0.322-0.324 on the HSV genome. This mutant adsorbed normally to the cell surface at the NPT, but failed to penetrate the cell membrane. Ts1204 appeared to bind to specific cellular receptors for HSV-1, since high multiplicities of infection of the mutant blocked subsequent superinfection of cells by HSV-1, but not by HSV-2. The penetration defect could be overcome either by brief incubation of ts1204-infected cells at the PT before temperature upshift to the NPT, or by treatment of the cells with polyethylene glycol, a compound which promotes fusion of membranes. Upon continued incubation of mutant virus-infected cells at the NPT, low numbers of capsids were assembled. Although these capsids contained some internal structure, they did not contain DNA. Another mutant, ts1208, lies in the same complementation group as ts1204. This mutant penetrated cells normally at the NPT but, like ts1204, assembled low numbers of capsids which did not contain DNA. Marker rescue experiments mapped the ts1208 lesion to the left of the ts1204 lesion, within BamHI u. The ability of high multiplicities of ts1204 to block superinfection of cells by HSV-1 but not by HSV-2 was utilised to determine the virus polypeptides involved in the recognition of specific cell surface receptors. A series of intertypic recombinant viruses, which induced both HSV-1- and HSV-2-specific envelope glycoproteins, were all found to be capable of penetrating cells previously infected with ts1204 at the NPT. This result suggests that the virus attachment complex may be composed of more than one glycoprotein, and that a mixture of both HSV-1 and HSV-2 glycoproteins are able to recognise and bind to cellular receptors specific for HSV-2, since no HSV-2 sequence was common to all recombinants. The alternative explanation that the recombinants contained undetected crossovers cannot, however, be ruled out. Ts1203 has a ts lesion which maps in a 450bp fragment located between mu 0.377-0.380 on the HSV-1 genome. This mutant assembled large numbers of capsids at the NPT, but failed to encapsidate DNA. In this respect ts1203 resembled ts1201, a mutant which lies in a different complementation group but also has a defect in packaging of virus DNA into capsids (Preston et al., 1983). At the PT, DNA encapsidation was less efficient in ts1203-infected cells than in wild-type virus-infected cells. In addition, the defect in ts1203 appeared to be irreversible upon temperature downshift in the absence of further protein synthesis. Greater than 99% of the DNA synthesized in both ts1203- and ts1201-infected cells at the NPT was endless, suggesting that the unpackaged mutant virus DNA remained in a concatemeric form. Complementation experiments, using other ts mutants which had lesions in HSV DNA fragment EcoRI f, showed that ts1203 represents a novel complementation group. In preparation for DNA sequencing experiments to determine the base change responsible for the ts1203 phenotype, the DNA fragment containing the ts1203 lesion, and the corresponding fragment from ts1203 rev-1, a spontaneous revertant of ts1203, have been cloned into plasmid vector pUC9. Preston et al. (1983) demonstrated that ts1201 failed to process the polypeptide p40 to its lower molecular weight forms at the NPT. This result, together with the earlier observations by Gibson and Roizman (1974), strongly suggested that p40 was involved in the virus DNA encapsidation process. In contrast to ts1201, however, ts1203 was found to process p40 correctly at the NPT. Immune electron microscopic experiments showed that p40 was associated with both ts1203 and ts1201 empty capsids at the NPT. Experiments were performed to investigate the reason for the failure of p40 to be processed correctly in ts1201-infected cells at the NPT. It was found that the high MW forms of p40 were translocated normally from the cytoplasm to the nucleus in these cells. It was also shown that p40 synthesized in cells infected with HSV-1 strain 17 syn+ was not phosphorylated to any detectable extent, in contrast to results obtained with other HSV-1 strains (Heilman, 1979; Braun et al., 1984). Thus, defects in either of these events are unlikely to be responsible for the failure of p40 to be processed correctly to its lower molecular weight forms in ts1201-infected cells at the NPT.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Virology
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-77334
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 09:11
Last Modified: 14 Jan 2020 09:11
URI: http://theses.gla.ac.uk/id/eprint/77334

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