<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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:596865 |
| Date | January 1997 |
| Creators | Bramley, J. C. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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