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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.

<I>Herpesvirus saimiri</I> (HVS) is a T-lymphotropic γ-herpesvirus of New World Monkeys. The entire genome of HVS has been mapped and shown to contain several open reading frames (ORFs) with striking homology to mammalian cellular proteins, suggesting that host genes have been captured during the course of evolution. Such a gene is ORF 15 that has 64% homology to human CD59 and 69% homology to CD59 from squirrel monkey, the natural host for HVS. CD59 is a membrane glycoprotein which inhibits cell lysis induced by the membrane attack complex of complement, an important component of the innate immune response. Therefore, its acquisition could provide the virus with a potent method for evasion of host immune defences. The aim of the study was to discover whether ORF 15 does indeed encode a functional protein. The sequence was expressed in insect cells using the baculovirus expression system. HVS CD59 expressing cells were found to be protected against lysis by human complement in comparison with cells infected with a control virus, demonstrating the presence of functionally active HVS CD59 on the cell surface. However, no cross-reactivity with antibodies to Hu CD59 was observed, despite the high degree of sequence homology. Therefore, a number of alternative approaches were explored to facilitate purification of the protein. The most successful of these proved to be creation of an epitope tagged molecule by inserting the FLAG octapeptide N-terminal to the main coding region. This has permitted the recognition of an 18 kDa molecule by an anti-FLAG monoclonal antibody in a Western blot under reducing conditions, opening the way for purification and characterisation of this novel protein.