Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603719
Title: Functional analysis of the transmembrane domain and cytoplasmic tail of Herpes simplex virus type-1 glycoprotein H
Author: Harman, A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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Abstract:
Recently a role for the transmembrane (TM) domain and cytoplasmic tail in mediating membrane fusion has been demonstrated in many virus fusion proteins and therefore the importance of these regions of HSV-1 gH in this process was investigated. Chimeric constructs were generated in which the TM domain and/or cytoplasmic tail of gH were replaced with analogous regions from other proteins and these constructs were characterised using two assays. Firstly a transient transfection cell fusion assay was used in which cells expressing HSV fusion proteins form syncytia with neighbouring untransfected cells. Secondly a complementation assay was used which measures the ability of these constructs to rescue the infectivity of a gH null virus. These experiments demonstrated the importance of both these regions of gH in mediating membrane fusion as judged by either assay. Specific sequence requirements within these regions were then examined by constructing a series of deletion, truncation or substitution mutants that were then tested using the same assay methods. The results confirmed and extended previous studies which had implicated an SVP motif in the cytoplasmic tail in membrane fusion, and identified key residues in the transmembrane domain (notably a central glycine residue) that are required for fusion function. No gH mutants were isolated that were functional in the cell fusion assay, yet failed to function in virus rescue assays. This implies that neither the cytoplasmic domain nor the transmembrane domain contains specific sequences required for assembly into the virus envelope. This was confirmed by showing that chimeric molecules in which both the TM and cytoplasmic tail domains of gH were replaced by the equivalent domains of the cell-surface protein CD8, were incorporated into virions as efficiently as a wild type gH molecule. It is unclear how gH molecules are directed into the virion envelope. Many mutants that were apparently non-functional in cell fusion were, nevertheless, capable of rescuing virus infectivity. More detailed analysis demonstrated, however, that in these instances the rescued virions entered cells much more slowly than normal virions and that the behaviour of mutant molecules in the two assays was generally consistent. It is apparent that rescue assays which merely record recovery of infectious virus can be misleading.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603719  DOI: Not available
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