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The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis. However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5� leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated. Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression. To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG. Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.

Apoptosis plays important roles in host defences against virus infection. It is therefore not surprising that viruses have developed a vast array of modulators that block this process at different stages within the apoptotic pathways. Intrestingly, Orf virus (ORFV), a member of the Parapoxvirus genus, did not reveal any of the known poxviral inhibitors of apoptosis, but was found to express a unique anti-apoptotic protein, ORFV125. The aim of this PhD project was to determine the subcellular localisation of this protein and to further characterise its anti-apoptotic activity. This included exploring its ability to inhibit early, intermediate and late events of apoptosis and identifying the mechanism by which this viral protein functions to prevent cell death. Experiments revealed that ORFV125 was localised to the mitochondria through a C-terminal mitochondrial-targeting motif, and this specific location was necessary for the protein�s anti-apoptotic function. Furthermore, the viral protein inhibited UV-induced apoptotic events at and downstream of the mitochondria such as cytochrome c release, caspase activation and DNA fragmentation. However, it was not able to prevent UV-induced activation of the c-Jun-NH₂ kinase (JNK), an event occurring upstream of the mitochondria, consistent with its localisation to this organelle. The ability to prevent apoptosis was comparable with that of the cellular anti-apoptotic protein Bcl-2, which belongs to a family of mitochondrial regulators of apoptosis. Although standard BLAST analysis failed to detect homology to anti-apoptotic members of the Bcl-2 family, a manual alignment of the primary sequence of ORFV125 with these proteins revealed characteristic residues of Bcl-2 homology (BH) domains within ORFV125. These motifs are conserved within the Bcl-2 proteins and important for their structure and function. In addition, mutating amino acids within the ORFV125 BH domains led to a loss of the anti-apoptotic function of the mutated proteins, indicating the functional importance of these residues for the viral protein. These observations suggest that ORFV125 might be classified as a viral Bcl-2-like protein. To provide evidence for this hypothesis, it was investigated if ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. The viral protein was able to entirely block the activation of the pro-apoptotic Bcl-2 family members Bak and Bax, although it did not directly bind to these proteins. Instead, ORFV125 interacted with a subset of the pro-apoptotic BH3-only proteins, which can trigger the activation of Bax and Bak. Furthermore, this study demonstrated that ORFV125 could inhibit apoptosis induced by BH3-only proteins to which the viral protein could bind. On the other hand, ORFV125 was not able to prevent the activity of pro-apoptotic proteins that it failed to interact with. This shows that ORFV125�s mechanism of action is to inhibit the activity of BH3-only proteins by binding and neutralising their function. Overall, these results provided evidence that ORFV125 is potent anti-apoptotic protein that can prevent UV-induced cell death without the participation of other ORFV proteins. Furthermore, the viral protein shared primary sequence and secondary structure similarities to Bcl-2 family members and acted in a Bcl-2-like manner to inhibit apoptosis.

The anaphase promoting complex (APC/C) is an ubiquitin ligase that is an essential regulator of multiple steps in the cell cycle. The complex consists of at least 12 subunits with a catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. The Parapoxvirus, Orf virus (OV), encodes a RING-H2 protein, B5L, with clear sequence similarities to APC11. The disruption of APC/C function leads to pre-mature entry into S phase and a delayed M phase exit and, potentially, apoptosis. This investigation explored the functional significance of the similarity between B5L and APC11 and specifically sought to determine if B5L manipulates cell cycle regulation by targeting APC/C function. Co-immunoprecipitation experiments from lysates of cells expressing a range of constructs revealed an interaction between B5L and APC2 in the same manner as seen with APC11. Furthermore, B5L was found to associate with endogenous APC/C. However, although APC11 promoted the formation of polyubiquitin chains in substrate-independent in vitro assays, B5L was inactive in this assay. Bioinformatics comparisons of APC11 and other known RING ubiquitin ligases with B5L and its poxviral homologues revealed some subtle differences. In particular a domain of APC11 (amino acids 61-74), that is essential for its ubiquitin ligase activity is not conserved in B5L or its homologues. When this APC11 domain was incorporated in place of the corresponding region of B5L (amino acids 59-67), the mutated B5L acquired ubiquitin ligase activity. On the other hand, APC11 protein in which the domain was replaced with that of B5L lost ubiquitin ligase activity. Stable cell lines expressing B5L showed an increased number of cells in G2/M phase (30�4%) compared with cell lines expressing APC11 (11�2%, n=3, p<0.05, ANOVA, Tukey�s), consistent with impaired APC/C function. APC/C substrates such as cyclin A, cyclin B and the thymidine kinase were stablized in B5L-expressing cells compared with control cells. Furthermore, transient hyper-expression of B5L induced apoptosis in 25�2% (n=3, p<0.05) of the cell population compared with only 6�1% apoptotic cells when APC11 was hyper-expressed. Analysis of the DNA content of OV-infected cells revealed enhanced DNA synthesis compared with cells infected with a B5L knockout OV. These observations indicate that B5L is a non-functional mimic of APC11. It associates with APC/C, but lacks ubiquitin ligase activity, and hence disrupts APC/C function. These abilities may enable OV to induce a cellular environment that enhances viral replication.

Includes corrigendum attached to back leaf. / Bibliography: leaves 114-132. / xvi, 132 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the complete nucleotide sequence and genome organisation of SCSMaV-RNA, examines the taxonomy of SCSMaV by phylogenetic analysis, and describes the development of diagnostic tests for application to field study. (abstract) / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2001

x, 123 leaves : ill. ; 26 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 Entomology, Waite Agricultural Research Institute, 1973

Typewritten copy. / "The embryoculture investigations and the results of the dissection of tomato spotted wilt virus infected seeds described in Section B were presented in my M.Sc. thesis in 1953. They are again included here for the sake of completion". / 85 leaves : ill. ; 26 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, 1957

xiii, 111 leaves : ill., graphs, tables, photos ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1976) from the Dept. of Plant Pathology, University of Adelaide

Ph.D. thesis 1970 from the Dept. of Plant Pathology, Waite Institute, University of Adelaide / iii, 97 leaves : ill., graphs / Title page, contents and abstract only. The complete thesis in print form is available from the University Library.

xiii, 111 leaves : ill., graphs, tables, photos ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1976) from the Dept. of Plant Pathology, University of Adelaide