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The aim of this study was to examine the mechanisms of transcription and replication of the paramyxovirus, simian virus type 5 (SV5). This was initially attempted using reverse genetics techniques and subsequently examining specific viral protein: protein interactions within the replication complex. A cDNA clone encoding a synthetic negative-sense RNA genome analogue was constructed. Reverse genetics techniques were used to attempt to characterise conditions which supported the transcription and replication of this genome analogue, with or without the use of wild-type helper virus but were unsuccessful. During the course of these studies, a number of mammalian cell lines inducibly expressing SV5 proteins were isolated. These cell lines were subsequently used to examine viral protein: protein interactions within the replication complex. When expressed alone, both P and V proteins exhibited diffuse cytoplasmic fluorescence and V was also found in the nucleus. However, when NP was expressed alone, it was seen as punctate and granular cytoplasmic fluorescence. The distribution patterns of the proteins changed when expressed in combination. Large cytoplasmic aggregates similar to those at late times in an SV5 infection were seen in cells which co-expressed NP and P. When NP was co-expressed with V, however, NP was partially redistributed to give diffuse cytoplasmic and nuclear fluorescence. This showed that both P and V proteins could interact with NP and suggested that V may play a role in keeping NP soluble prior to an ordered encapsidation process. Extracts from these cell lines were then used in a novel protein: protein capture assay and demonstrated that NP could interact with both P and V proteins. NP expressed by the cell line was shown to contained both soluble and polymeric forms of NP. P was shown to bind both forms of NP, while V could only bind soluble NP. Since P and V proteins are amino co-terminal, the site of interaction between P and polymeric NP was predicted to be in the P unique C-terminus. This was strengthened when a P-specific C- terminal mAb was found to block the binding of P with polymeric NP. Deletion mutant analysis in the C-terminus of the P protein showed that the mAb binding site was at the extreme C-terminus of the protein suggesting this is the point of interaction between P and polymeric NP. Possible roles for these protein: protein interactions and implications for the paramyxovirus replication complex are discussed.

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Copy of author's previously published article on back end-paper. / Bibliography: leaves 105-158. / xii, 160, [58] leaves, [35] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the early events in the synthesis of HIV RNA and integration if viral DNA using a cell-to-cell transmission of infection model. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology & Immunology, 1998

Reciprocal interference experiments between Potato virus Y (PVY) and Pepper mottle virus (PeMV) in Capsicum annuum L. 'Tabasco' and 'Special pepper' (a selection of Anaheim chilli peppers), showed suppression of local lesion production in both directions but incomplete suppression of challenge virus replication (incomplete cross protection). However, suppression was reduced by increasing the concentration of the challenge virus. The source of inoculum of the viruses did not have a significant effect on the subsequent interference between the viruses. A direct relationship was established between counts of local lesions and virus particles counted using the electron microscope. Tobacco etch virus (TEV) was transmitted from one Capsicum annuum L. 'Tabasco' plant to another in the same pot within 4 days after infection of the test plant. Transmission probably took place via root grafts, as the necrotic roots of the test plants intertwined with the uninoculated Tabasco plants. Mixed infections of PVY and PeMV resulted in the production of PVY-N, a new strain of PVY. The new strain, which could be recognized by changes in biological and serological properties, was produced only in mixed infections and was stable after six serial transfers in several hosts. Evidence suggests that the development of the new strain is host dependent. Potato Virus Y in mixed infections with PeMV or TEV in C. annuum L. 'Anaheim' did not induce local lesions, systemic necrosis nor death of Special pepper, while PeMV alone induced the death of Tabasco. The behavior of TEV in Tabasco in a mixed infection was temperature dependent, while TEV alone induced wilt and death of Tabasco irrespective of the greenhouse temperature and season of the year.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1979. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 142-145. / Ph.D.

Copy of author's seven page article in pocket inside back cover. / Bibliography: leaves 118-143. / x, 143, [52] leaves, [23] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

Bibliography: leaves 94-104

1,10-phenanthroline and its non-chelating isomer, 1,7-phenanthroline were used to inhibit the replication of vaccinia virus (VV). Serial passage of VV in the presence of various concentrations of either 1,10-phenanthroline or 1,7-phenanthroline was carried out. No drug resistant mutants were isolated, suggesting that the observed inhibition was due to a cellular protease as opposed to the putative viral protease G1L. Cultures infected in the presence of the inhibitors, were radio labeled with �����S-methionine at various time points post infection, to determine which step of VV replication was inhibited. Infections in the presence of 1,10-phenanthroline proceeded only through early gene transcription, suggesting that the point of inhibition was uncoating. Finally, cells infected with VV with or without the inhibitors at time zero and eight hours post infection were used to generate transmission electron microscopic images. Taken together these results indicate that inhibition was occurring at the level of uncoating. / Graduation date: 1999

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy