Abstract
Hepatitis C virus (HCV) RNA replication occurs in the cytosol of infected cells within a specialised membranous compartment. How the viral non-structural (NS) proteins are associated and organised within these structures remains poorly defined. We employed a super-resolution microscopy approach to visualise NS3 and NS5A in HCV infected cells. Using single molecule localisation microscopy, both NS proteins were resolved as clusters of localisations smaller than the diffraction-limited volume observed by wide-field. Analysis of the protein clusters identified a significant difference in size between the NS proteins. We also observed a reduction in NS5A cluster size following inhibition of RNA replication using daclatasvir, a phenotype which was maintained in the presence of the Y93H resistance associated substitution and not observed for NS3 clusters. These results provide insight into the NS protein organisation within hepatitis C virus RNA replication complexes and the mode of action of NS5A inhibitors.