| Summary: | The research on hepatitis C virus protein NS5A has developed rapidly over the decades, primarily with the advent of the JFH1 cell culture infectious clone which allowed the study of all aspects of the virus lifecycle from entry to exit. As the important target of DAAs, the NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, a mutagenic study of 12 absolutely conserved and surface-exposed residues were conducted within the context of a JFH1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. Whilst P35A exhibited a modest reduction in infectivity, V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high-resolution confocal microscopy, it was demonstrated that V67A and P145A disrupted the recruitment of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken collectively, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. In parallel, this study also set out to investigate the interactions of NS5A domain I with cellular proteins by the approach of quantitative proteomic analysis. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.
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