Functional interactions of the adenovirus serotype 5E4orf3 protein
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.