| Summary: | Hepatitis C virus causes a chronic infection in ~3% of the world’s population and is a leading cause of liver diseases such as cirrhosis and hepatocellular carcinoma. It is a positive-sense single-stranded RNA virus that persists in ~85% of infections. Viral genome replication occurs within a specialised membranous compartment, termed replication factories. This provides an environment suitable for the production of infectious virus, and correct formation and maintenance is critical for virus replication. The process is coordinated by the non-structural proteins in a macromolecular protein assembly, but the precise mechanisms of biogenesis and protein organisation within replication factories are unknown. New super-resolution light microscopy approaches allow resolutions of tens of nanometres, 10-fold higher than standard wide-field or confocal microscopy. The goal of this research was to use these techniques to determine the organisation and architecture of proteins within replication factories. Super-resolution imaging revealed clusters of viral proteins that were equivalent to the diffraction limited puncta observed by wide-field microscopy. A detailed analysis of protein clusters identified significant differences in size and organisation between the non-structural proteins NS3 and NS5A with a defined minimum distance to the cluster centroid. Additional investigations into the functions of NS5A revealed altered cluster phenotypes with both pharmacological inhibition and mutants defective in phosphorylation. A number of strategies were also explored to facilitate fluorescence labelling of viral components in replication factories. In parallel, investigations into the biogenesis of replication factories were explored by characterising interactions between hepatitis C virus and autophagy. This study identified a requirement of HCV replication for early steps in the formation of autophagosomes. The findings from this research are the first descriptions using super-resolution microscopy to understand the hepatitis C virus replication complex and provide insight into the organisation and architecture of the non-structural proteins during infection.
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