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In this study two enteric viruses, poliovirus and adenovirus type 40, were used to investigate virus - host cell interactions in the gastrointestinal environment. Human enteric adenoviruses have been previously shown to be sensitive to IFN-treatment of cells and a STAT-deficient cell line allows improved growth of enteric adenovirus type 40. Two adenoviral gene products, the virus-associated RNA (VA RNA) and the El A, have been previously shown to be responsible for viral countermeasures against IFN actions in the host cell.

In recent years, interest in the cellular structures known as PM.l Oncogenic Domains (PODs) has been growing, as it has become increasingly clear that these structures may be implicated in several cellular functions, such as the regulation of the levels of active proteins in the nucleus, transcriptional regulation, suppression of growth and transformation, antiviral response, cell-cycle regulation and apoptosis. The PODs constitute large, multi-protein complexes associated with the nuclear matrix, and are visualised under immunofluorescence analysis as discrete dots numbering 10 to 20 per nucleus. A major protein component of the PODs, the promyelocytic leukaemia protein (PML), appears to be central to the function of these nuclear bodies. Notably, disruption of the PODs is observed in several malignant tissues, at the beginning of mitosis, and during infection with several viruses. During the course of a wild-type adenovirus infection, the PML protein is redistributed from the normal punctate, nuclear bodies. inr.o "track-like" structures, which colocalise with the viral protein, E40rf3. By ide itifying a mutant adenovirus defective in track formation, it was possible to ass"gn responsibility for POD reorganisation to this single viral gene product. Western blotting analysis of PML has demonstrated the existence of a characteristic pattern of PML isoforms, some apparently modified by the ubiquitinlike protein, Sl. MO-I. Analysis of the PML species present in adenovirus infected cells has shown that this characteristic pattern is altered, with the loss of SUMO-Imodified isofon 11.3, and the appearance of a novel, infection specific band. This thesis de ..cribes attempts to further investigate the nature and functional significance of the interaction between the viral E40rf3 protein and the cellular protein, PML, as occurs during adenovirus infection, by expressing Orf3 and mutants of Orf3 in eukaryotic cells through the use of plasmid based gene expression systems. By these methods, Orf3 was confirmed as necessary and sufficient for the redistribution of PML from the PODs to the "track-like" structures associated with adenovirus infection, and regions of the Orf3 protein implicated in the nuclear retention of the protein or protein stability were identified. Further, a potential link between POD redistribution and the adenovirus infection-specific PML isoform was investigated using Western blotting analysis on cell lysates derived from eukaryotic cells expressing Orf3 or mutants of Orf3 in isolation from other viral compnents. To facilitate these investigations, attempts were made to develop cell lines capable of permanent exp« ~:;10nof Orf3 protein. These experiments led to the identification of a potentially cytc pathic effect associated with the long-term expression of Orf3 in eukaryotic cells. Attempts were also made to construct whole, infectious virus, expressing mutant Orf3 proteins.

Replication defective adenoviruses have emerged as promising vectors for delivery of vaccine antigens. The development of new vaccine vectors has recently focused on serotypes t, which the human population is less exposed in order to circumvent pre-existing anti vector immunity. This thesis describes the construction and optimisation of ChAdOX1, a new vector based on chimpanzee adenovirus Y2S, which has recently been manufactured to clinical grade for a Phase 1 human trial. Comparative immunogenicity studies between vectors of different serotype were performed in mice, with careful consideration of the infectious titer of vector preparations, since this parameter can confound studies based solely on viral particle estimation. Aft intramuscular administration, HAdV-S (Human adenovirus C) based vectors elicited superior transgene product specific T cell and antibody responses compared to a selection of chimpanzee adenovirus vectors (from Human adenovirus EJ including ChAdOX1. The construction of ChAdOXl in a bacterial artificial chromosome (BA C), enabled precise, and flexible modification of the genome by recombmation mediated genetic engineering. (recombmeering). Reverse genetics was performed to identify vector determinants of immunogenicity. Chimeric ChAdOXl vectors were created by replacement of native virus associated RNA (VA-RNA) and fiber sequences with the corresponding sequences from HAdV-5 Using these chimeric vectors, the importance of innate immunity and vector transduction in determining vector immunogenicity was investigated. Though the mechanisms responsible ultimately remain unclear, superior transgene product specific immune responses with HAdV-5 correlated with higher levels of transgene expression in vivo after vector administration. The current study has conclusively demonstrated that neither VA-RNA sequences, nor the fiber protein, are responsible for differences in immunogenicity between vectors, contrary to hypotheses based on previous studies.