Herpes virus evasion of the interferon-alpha-stimulated immune response

Interferon (IFN) is a cytokine that acts rapidly via the Jak/Stat signalling pathway to induce an antiviral state in uninfected cells, and cell death in those already infected. Many viruses have evolved mechanisms to antagonize the IFN system, targeting all the major components involved in receptor binding and signalling. While a number of these viral inhibitors are homologous to cellular proteins involved in IFN down-regulation (e.g. vIRFs), many share little resemblance to known proteins. To determine the IFN-blocking properties of these proteins, functional protein-protein interaction studies are required. The first element of this study addressed the development of a new and rapid functional screening method. Based on inherent properties of the 2fTGH cell line, the system used selection upon a construct containing the guanine phosphoribosyl-transferase gene, tightly regulated by the 6-16 gene IFN-stimulated response element. By addition of 6- thioguanine in the presence of IFN, cells containing a functional pathway would die from toxic metabolites, however those with an inhibited pathway would survive. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN@ and 6-thioguanine resulted in the outgrowth of cells that were no longer responsive to IFN@. It was also demonstrated that selection occurs if members of the Jak-STAT signalling pathway were lost. Furthermore, the screening system was validated using a known suppressor of IFN signalling, the human papillomavirus E7 gene. Expression of the E7 gene product resulted in loss of the ability of 2fTGH cells to respond to IFN@ treatment due to a functional disruption of the signalling pathway, and thus substantiated the suitability of the assay. Following development of the functional screen, the approach allowed investigation into the Herpes Simplex Virus 1 (HSV1) for potential gene products involved in IFN@ signalling inhibition. This second part of the study involved the construction of a primary library from HSV1 genomic DNA and introducing it into the 2fTGH functional screen. Using a shotgun cloning approach, the library could be quickly prepared and still represent the greater majority of viral genes. Consequent selection with IFN@ and 6- thioguanine resulted in the outgrowth of cells, producing a number of clones that were no longer responsive to IFN@. Each clone was confirmed to have impairment in the IFN pathway by way of the integration of viral genes rather than some spurious mutation and maintained under selection to discourage reversion. Identification of the causative viral gene in each clone appeared to be more difficult than first expected. Following a number of different tactics, a PCR screen was established that allowed correlative detection of the viral genes present in each selected clone. This gave rise to a number of HSV1 genes potentially implicated in the inhibition of IFN@ signalling, namely the UL24, UL28, US6 and US9 genes. The third section of this study concentrated on the elucidation of the precise proteinprotein interplay of these implicated viral genes on the components of the Jak-STAT signalling pathway. Despite a variety of cloning approaches, only the US9 gene was successfully inserted into an expression vector and further examined. Re-selection of this positive clone in the 2fTGH system suggested a defined inhibitory role of the US9 gene product, as indicated by the disruption of signalling molecules. It appears that this function may be carried out indirectly or through some shuttling mechanism yet to be understood, as interactions of the US9 protein with Jak-STAT elements were suggested to be a possibility yet were inconclusive. This study has demonstrated that through the development of an IFN-responsive functional screen, entire viral genomes can be scanned for novel viral genes involved in IFN@ signalling. As certain difficulties affect viral gene identification, the current screening model may be more suitable for resolving the function of individual genes however a few adjustments to the preparation of genetic libraries could facilitate more efficient library screening. Consequently, the system could be extended to examine any virus, enabling IFN-resistance strategies to be consolidated, and development of a more complete regime of anti-viral compounds