Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.

Norovirus (NoV) and Sapovirus (SaV) are major causes of outbreak gastroenteritis worldwide. NoV and SaV are highly infectious, have multiple transmission routes and have a short incubation period, thereby facilitating rapid intercontinental spread of new variants. Consequently, a treatment would be advantageous for controlling them. However, currently little is known about the replication cycle and evolution of human NoV or SaV as neither are culturable. NoV and SaV are RNA viruses of the Caliciviridae family and have great genetic diversity which is thought to facilitate irnmune evasion. Consequently variants of NoV GI1.4 arose in 1996, 2002, 2004 and in 2006 and resulted in pandemics. Therefore, in this study, the role of the two main mechanisms associated with generating viral diversity; recombination, and point mutation were investigated for NoV and SaV. Physiological and kinetic properties of three NoV RdRps (genotypes, Gll.b, Gll.4, Gll.7) and two SaV RdRps (genogroups GI, GII) were also investigated. RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the Calicivirus genus Norovirus (NoV) has led to a rise in the identification of NoV recombinants and they are now reported at high frequency. Despite this no classification system exists for recombinant NoVs As a result, there is duplication in reporting novel recombinants and the precise number of novel NoV recombinant types is unknown. Therefore, in order to elucidate thero!e of recombination in NoV evolution, 121 NoV nucleotide sequences, compiled from the GenBank database and published literature, were analysed for recombination events. NoV recombinants and their recombination breakpoint were identified using three methods: phylogenetic analysis, Simplot analysis and the Maximum Chi-Squared method. In total 19 unique NoV recombinant types were identified in circulation across the globe and they had a common recombination point near the ORF1/2 overlap. Recombination at the ORF1/0RF2 overlap could have important implications in NoV evolution as it enables a virus to swap its antigenic determinates (capsid) and thereby avoid immune clearance in an analogous manner to antigenic shift in influenza virus. This study also examined the role of NoV and SaV replication in generating viral diversity by comparing the physiological, kinetic and biochemical properties of five genotypically distinct RdRps from two different genera of the Caliciviridae. Genetically diverse HuCV RdRps were expressed in Escherichia coli and characterised in an in vitro assay designed for this study. The results indicated that despite high sequence variation between the five enzymes (between 6% and 71% amino acid difference) they shared similar physiological properties. Though there was some variation in their template usage and kinetic properties. SaV was able to perform primer dependent replication on homopolymeric A RNA whereas the NoV RdRps were not. Additionally, NoV RdRps had a higher incorporation rate and were more kinetically efficient than the two SaV RdRps. The incorporation fidelity of the five enzymes was similar (between 2.2x10-5 to 8.9x10-4 ), although interestingly the most prevalent strain, Gll.4, had the lowest fidelity of the caliciviruses. Therefore, suggesting that RdRp fidelity has an important role in NoV evolution. Overall, this study illustrated that NoV and SaV generate genetic diversity in a similar fashion to other RNA viruses, that is, a delicate combination of recombination, point mutation and replication efficiency. Understanding the mechanisms involved in viral replication and genomic diversity of the calicivirus RdRps is essential if a successful control strategy for the human caliciviruses is going to be developed.

Bibliography: leaves 127-161. / x, 161, [94] leaves, [30] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Direct transmission from infected cells to uninfected cells, otherwise known as cell-to-cell infection, is one mode of viral spread adopted by HIV-1. Transcriptional aspects of HIV-1 replication were examined following cell-to-cell infection. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997

Bibliography: leaves 94-104 / viii, 104, [78] leaves, [19] 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 Biochemistry, 1984

Tetraviruses are a family of small non-enveloped positive sense RNA viruses that exclusively infect members of the order Lepidoptera. Their replication biology is poorly studied because, with the exception of Providence virus (PrV), tetraviruses are unable to replicate in tissue culture cells. The overall aim of the research described in this thesis was to develop a fundamental understanding of the replication of tetraviruses, focussing on the site of replication within host cells and in particular, the subcellular localisation of the viral replicase. Helicoverpa armigera stunt virus (HaSV, Genus: Omegatetravirus) was chosen for this study because it is the only tetravirus for which the cDNAs have been shown to be infectious. In the absence of tissue culture cell lines susceptible to HaSV infection, the approach was to use confocal fluorescence microscopy to examine the subcellular localisation of the HaSV replicase fused to enhanced green fluorescent protein (EGFP) in mammalian and insect tissue culture cells. The replicase (with EGFP fused at its C-terminus) localised to punctate structures throughout the cytoplasm of transfected HeLa and Sf9 cells. These structures were then shown – using live cell imaging and time lapse photography – to behave similarly to cellular endocytic organelles and fluorescence partially overlapped with membranes containing the late endosomal marker protein CD63. Biochemical fractionation of Sf9 cells expressing the replicase via a recombinant baculovirus (as well as transfected HeLa and Sf9 cells expressing EGFP-replicase fusion proteins) demonstrated that the replicase was strongly associated with detergentresistant membranes (DRMs) in these cells. Deletion analysis of the replicase coding sequence revealed two regions involved in the generation of the punctuate structures. Firstly, the C-terminal half of the replicase RNAdependant RNA polymerase domain was found to be essential for targeting and the tight association with DRMs while the second region, within the Nterminal 44 amino acids, enhanced localisation through a combination of secondary structural elements and sequence-specific functions. A comparative immunofluorescence study on PrV, which replicates as a persistent infection in an insect midgut cell line, showed that the PrV replicase also localised to punctate structures in the cytoplasm. Biochemical fractionation showed that the replicase was also strongly associated with DRMs. This thesis describes the development of new experimental systems for the study of tetravirus replication biology and the data lead to the conclusion that the HaSV replicase associates with DRMs derived from alternate endocytic pathway organelles.

The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.

The influenza A virus genome codes for up to 12 proteins. Segment 2 encodes three proteins, the polymerase subunit PB1, a small protein PB1-F2 and an N-terminally truncated version of PB1 called N40. Different functions have been reported for PB1-F2 such as induction of apoptosis, regulation of the viral polymerase activity, enhancement of secondary bacterial infections and modulation of the innate immune system. So far, no function has been ascribed to N40. To study PB1-F2 in more detail, its coding sequence was deleted from its original position and inserted downstream of the PB1 (segment 2), NA (segment 6) or M (segment 7) open reading frames (ORF) employing different strategies, including the use of an overlapping Stop-Start cassette, a duplicated promoter sequence and the self-cleaving 2A peptide derived from foot-and-mouth disease virus. Viruses with bicistronic segments were rescued and tested for their ability to express PB1-F2. Whereas no expression of PB1-F2 was detected from bicistronic segments 2 and 7, expression of PB1-F2 from segment 6 was observed in high levels. However, the phenotype of all these viruses was similar to that of viruses lacking PB1-F2 which made mutational analysis of PB1-F2 not worthwhile. Previously, the function of PB1-F2 was mainly studied using a virus deficient in PB1-F2 production but showing increased N40 expression. In the present study, recombinant WSN viruses lacking either PB1-F2 or N40, or both proteins were engineered and the effects of these mutations on the viral life cycle were examined. Viruses deficient for PB1-F2 that overexpressed N40 showed the most attenuated phenotype, whereas the loss of PB1-F2 alone did not obviously affect virus replication. Reduced viral polymerase activity was observed for viruses lacking N40, however attenuation in vivo was only seen in combination with the loss of PB1-F2. Neither the loss of PB1-F2 nor N40 alone had a great impact, but changes in the expression level of both proteins were disadvantageous for the virus. Increased levels of N40 shifted the polymerase activity towards replication, suggesting a new function for N40. Thus, it was shown that the segment 2 gene products and their expression level influence viral replication and pathogenicity, and a careful design of mutant recombinant viruses is vital for determining the experimental outcome.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

This research project deals with the characterization of self-replicating Kirsten murine viral DNA. The replication of this viral DNA and tetracycline resistance conferred to bacteria by this viral DNA will be studied. The restriction endonuclease and Southern blot analysis revealed a fragment of pBR322 from the Hind III and Pst I site that is located in the 3' end of the MLV-K:E molecule. Single stranded sequencing of the two terminal ends of this fragment verified that the 3' end of MLV-K:E contains identical sequence homology to pBR322. The presence of this pBR322 fragment explains the unusual properties of the MLV-K:E molecule. However, tetracycline resistance is less in E. Coli containing MLV-K:E than E. coli containing pBR322 as determined by zone of inhibition assay. This may be due to alteration in the promoter region of the tetracycline gene.

"February, 2002" / Includes bibliographical references (leaves 156-182) / xiv, 182, [26] leaves : ill. (some col.) ; 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 Molecular Biosciences, 2002