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Hepatitis C virus NS5A is a multi-functional viral protein essential for viral replication and assembly, although the exact role the protein plays in the viral lifecycle remains unclear. A vast array of functions have been attributed to NS5A in recent years, despite the lack of enzymatic activity. NS5A has been found to interact with over 130 host proteins including many which are central to cellular signaling pathways. NS5A is composed of three domains separated by regions of low complexity. All three domains perform important functions in the viral lifecycle. Domains I and II are essential for viral replication whereas domain III is required for viral assembly. However, the role that NS5A and its individual domains may play in modulating viral translation remains controversial. Previous studies have utilized translation reporter systems that do not accurately reflect the role of the viral 3´-UTR in modulating viral translation. We and others have shown that NS5A binds to the poly-U/UC region of the 3´-UTR. In addition to serving as the initiation site for negative strand synthesis the 3´-UTR functions to significantly enhance viral translation. The mechanism of translation enhancement remains unclear but may involve long range RNA-RNA interaction with the IRES, the binding of cellular proteins which stimulate translation and/or the recycling of ribosomes. Therefore, the protein-RNA interaction between NS5A and the poly-U/UC region has the potential to modulate viral translation. Therefore we set out to determine the role of NS5A and its individual domains in modulating viral translation and the role of the NS5A-poly-U/UC region interaction in this modulation. Utilizing monocistronic RNA reporters which contain the viral 5´- and 3´-UTRs and an internal Renilla luciferase reporter gene, we determined that NS5A specifically down-regulates viral translation in a dose-dependent manner through a mechanism dependent upon the presence of the poly-U/UC region in the viral 3´-UTR. Furthermore, we have re-tested the effect using full-length HCV genomic RNA reporters. These results suggest that NS5A is able to interfere with the stimulation of viral translation exerted by the 3´-UTR. This down-regulatory function of NS5A may function in mediating a switch from translation to replication, a step required in the lifecycle of a positive sensed RNA virus. Having established a role for NS5A in modulating viral translation, we then aimed to determine which region of NS5A was responsible for this effect. We found that each of NS5A domains was capable of this modulatory effect on viral translation independently. Although surprising, this finding is not entirely unexpected as each domain has been shown to retain the ability to bind to the poly-U/UC region. Within NS5A domain I we identified a 61 aa. region sufficient for translation down-regulation. Furthermore, we have identified a number of positively charged residues within this region involved in the modulation of viral translation, in particular arginine 112 (R112). This residue has previously been found to be at the domain I dimer contact interface and to form an intermolecular hydrogen bond with glutamic acid 148 (E148). We found that mutations R112A and E148A individually negate the ability of domain I to modulate viral translation and these mutations impede the formation of domain I dimers. Additionally, the R112A mutation appears to affect the ability of domain I to interact with the poly-U/UC region of the viral 3´-UTR alluding to the possible mechanism of translation modulation. Finally this mutation was lethal in an HCV subgenomic replication, confirming the link between NS5A dimerization, RNA binding and viral replication. These results collectively point to a crucial role for the NS5A arginine 112 residue in the modulation of HCV lifecycle by NS5A. Within NS5A domain II, we identified a 47 aa. region sufficient for translation modulation. Through the mutation of positively charged amino acids within this region, we found that lysine 312 (K312) was essential for this effect. The ability of this domain to modulate viral translation was completely lost when K312 was mutated within a full domain II protein fragment. The mechanism behind this modulation remains unclear but the 47 aa. region identified has been previously found to contain a region proposed to make contact with poly-U RNA and the K312 residue was suspected to interact directly with such RNA. Furthermore, this region interacts with the host protein cyclophilin A, an interaction that enhances the RNA binding ability of domain II. These findings strongly suggest that domain II modulates viral translation by binding within the poly-U/UC region. While investigating the modulation of viral translation by NS5A domain III we determined that the C-terminal 31 aa. are sufficient for the effect of this domain on viral translation. Through alanine scanning mutagenesis we identified glutamic acid 446 (E446) as playing a key role in the modulatory function of this region. Within a domain III protein fragment mutation of this E446 residue abolishes the modulatory function of this domain towards HCV translation. The mechanism behind this modulation and the role of E446 in this effect remains to be determined. These findings suggest that in addition to being essential for viral replication and assembly, NS5A has an important role in modulating viral translation through a mechanism requiring the poly-U/UC region of the viral 3´-UTR. Furthermore, each domain of NS5A appears to contribute to this effect. These results support the description of NS5A as a multi-functional protein and the further characterization of its functions may aid in the development of novel antivirals targeting the numerous functions of this complex, and at times puzzling, viral protein.

The development of specific antiviral drugs directly targeting the hepatitis C virus (HCV) is clinically important, as the current standard interferon/ribavirin combination treatment is only partially effective, expensive and often associated with severe side effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternative or additional therapy. To date, the development and in vitro evaluation of PIs is restricted to the genotype 1/2 based replicon and the genotype 2a full length viral cell culture system. However, proteases of the different HCV genotypes vary substantially in their amino acid sequence and secondary structure and require separate evaluation of their efficacy before they go into clinical trials. To address this issue, a panel of intra- and intergenotypic recombinants based on the recombinant infectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. The viability of these recombinants was assessed in the Huh7.5 cell culture system, where replicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinants containing genotype 1a, 1b, 3a, 4a and 6a derived proteases were replication defective, whereas the recombinant with genotype 5a derived protease replicated efficiently after acquiring cell culture adaptive mutations. The replacement of not only the NS3 protease gene region, but also its cofactor NS4A, allowed the generation of replication competent intra- and intergenotypic recombinants for all 6 major genotypes. Replacing the NS3 protease of the recombinants with that of patientderived proteases also generated replicating recombinants, greatly expanding the panel of intergenotypic recombinants available for phenotyping and PI evaluation. However, intra- and intergenotypic recombinants showed substantial differences in their replication kinetics, which may be influenced by naturally occurring polymorphism between genotypes and the differential requirement of adaptive/attenuating cell culture mutations. Genotype 1a recombinants replicated very poorly, which may be due to incompatibilities between the type 1a NS3/4A protease and the type 2a backbone. 50% inhibitory concentrations (IC50) of different PIs were measured using Foci Forming Units/ml (FFU/ml) reductions and replication inhibition assays. The different recombinants showed consistent, genotype-associated differences in their susceptibility to the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showing approximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants. These observations are consistent with major differences in response rates found in recent treatment trials of genotype 1, 2 and 3 infected patients. Differences in susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derived recombinants being twice as susceptible than genotype 3a, 4a and 5a derived recombinants. Passaging the intra- and intergenotypic recombinants under increasing concentrations of PI allowed the identification of PI resistance mutations. Resistance mutations to BILN 2061 mapped to the previously identified positions 156 and 168 within the NS3 protease, with a great diversity of amino acid substitutions observed within each genotype. Reintroduction of the identified resistance mutations into the original recombinant viruses conferred increased resistance towards BILN 2061 and some mutations also affected replication kinetics of the recombinants. The developed system will be of major value for the phenotypic characterisation of naturally occurring and treatment induced resistance mutations within all 6 major HCV genotypes towards different PIs. This will allow treatment response predictions for newly developed PIs before they enter clinical trials and the development of individually tailored antiviral treatment regimes.

The viral dynamics of the hepatitis C virus (HCV) in newly acquired infection are not well understood. HCV exists within an individual as a spectrum of minor variants termed quasispecies. The evolution of minor variants may contribute to viral escape of the host?s immune response, thereby facilitating development of chronic infection. The hypervariable 1 region (HVR1) is the most heterogeneous part of the HCV genome and contains a putative B-cell epitope. Thus, diversity in HVR1 could be a strategy used to evade neutralising antibodies. Acutely infected individuals (n=24) were examined with the aim of defining HVR1 quasispecies diversity in acute infection. The characterisation of the E1/HVR1 sequence and host specific evolution of HCV minor variants in treatment nonresponders was also investigated. HCV E1/HVR1 fragments were amplified from 48 sera using a combined reverse transcription-polymerase chain reaction (RT-PCR). Products were TA cloned into pCRIITOPO and approximately 10-20 clones were sequenced from each sample. HVR1 quasispecies diversity was examined longitudinally via sequence analysis. Quasispecies diversity was characterised primarily by mean nucleotide diversity. The mean HVR1 diversity of the acute cohort (n=48; 2.12% ?? 2.22) was lower than the diversity obtained for a cohort of chronically infected individuals (n=99; 4.5% ?? 5.1). There was no significant difference in mean HVR1 diversity between the HIV/HCV co-infected and HCV mono-infected groups (p=0.99) or between the clearer and non-clearer groups (p=0.85). Examination of amino acid usage and the hydropathic profile of each position in HVR1 revealed that sequence variation was confined to specific sites. The investigation of host specific evolution of HVR1 quasispecies demonstrated that minor variants (comprising 10- 20% of a population) became the dominant species over time in two treatment non-responders. These variants bore mutations that were not reflected in the consensus sequence of their respective populations at the initial timepoint analysed. Common infection was identified by 98% HVR1 sequence homology within two pairs of individuals. The evolution of common strains appeared to be different between individuals, suggesting host pressures may influence quasispecies evolution. This thesis provided an insight into the viral dynamics and host specific evolution of acute phase quasispecies.

Includes five copies of author's previously published articles in back pocket. / Bibliography: leaves 120-142. / xvi, 142, [59] leaves, [25] leaves of plates : ill. (some col.) ; 35 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines HCV-specific (Hepatis C virus-specific) protein and RNA expression in liver tissue from anti-HCV positive patients. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The viral dynamics of the hepatitis C virus (HCV) in newly acquired infection are not well understood. HCV exists within an individual as a spectrum of minor variants termed quasispecies. The evolution of minor variants may contribute to viral escape of the host?s immune response, thereby facilitating development of chronic infection. The hypervariable 1 region (HVR1) is the most heterogeneous part of the HCV genome and contains a putative B-cell epitope. Thus, diversity in HVR1 could be a strategy used to evade neutralising antibodies. Acutely infected individuals (n=24) were examined with the aim of defining HVR1 quasispecies diversity in acute infection. The characterisation of the E1/HVR1 sequence and host specific evolution of HCV minor variants in treatment nonresponders was also investigated. HCV E1/HVR1 fragments were amplified from 48 sera using a combined reverse transcription-polymerase chain reaction (RT-PCR). Products were TA cloned into pCRIITOPO and approximately 10-20 clones were sequenced from each sample. HVR1 quasispecies diversity was examined longitudinally via sequence analysis. Quasispecies diversity was characterised primarily by mean nucleotide diversity. The mean HVR1 diversity of the acute cohort (n=48; 2.12% ?? 2.22) was lower than the diversity obtained for a cohort of chronically infected individuals (n=99; 4.5% ?? 5.1). There was no significant difference in mean HVR1 diversity between the HIV/HCV co-infected and HCV mono-infected groups (p=0.99) or between the clearer and non-clearer groups (p=0.85). Examination of amino acid usage and the hydropathic profile of each position in HVR1 revealed that sequence variation was confined to specific sites. The investigation of host specific evolution of HVR1 quasispecies demonstrated that minor variants (comprising 10- 20% of a population) became the dominant species over time in two treatment non-responders. These variants bore mutations that were not reflected in the consensus sequence of their respective populations at the initial timepoint analysed. Common infection was identified by 98% HVR1 sequence homology within two pairs of individuals. The evolution of common strains appeared to be different between individuals, suggesting host pressures may influence quasispecies evolution. This thesis provided an insight into the viral dynamics and host specific evolution of acute phase quasispecies.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Thesis (Ph.D.)--University of Western Australia, 2006.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.