HCMV encodes a plethora of immune-modulating functions, many of which have yet to be assigned to specific genes. In prospect of performing high-throughput screens to identify and characterise such functions, a library of recombinant adenoviruses (RAds) each encoding a V5 epitope-tagged HCMV protein was generated. Protein expression was validated and characterised for the vast majority of RAds by western blot and immunofluorescence. HCMV has been reported to both upregulate cell surface expression of Fas, and render cells resistant to Fas-mediated killing. This thesis demonstrated that Fas levels are markedly reduced at the surface of HCMV-infected cells as an early function that persists through the late phase. Screening a panel of HCMV deletion mutants eliminated 83 genes as not required for Fas downregulation, while screening the RAd library did not identify any single HCMV gene as being sufficient for this function. Deep sequencing of the HCMV transcriptome recently led to the identification of UL150A as a novel protein-coding gene. To test this prediction, UL150A was tagged within the strain Merlin genome. UL150A was shown to encode multiple protein products, and be expressed with early and late kinetics. In a screen of the RAd library, gpUL4 was observed to be secreted from cells. To investigate this function in the context of HCMV infection, an epitope-tag was inserted at the 3’-end of the UL4 gene in the strain Merlin genome. Tagged gpUL4 was secreted from cells infected with strain Merlin. Secreted gpUL4 was more heavily glycosylated, and produced in greater abundance than its intracellular counterpart late in infection. Active secretion would be consistent with gpUL4 acting as a virokine, cytokine or cytokine/chemokine-binding protein. gpUL4 purified from supernatants of Merlin- or RAd-UL4-infected cells inhibited NK cell degranulation. Furthermore, gpUL4 did not copurify with virus particles, indicating it is unlikely to be a virion component.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:569935 |
| Date | January 2012 |
| Creators | Seirafian, Sepher |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/46644/ |
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