A key role for hepatitis C virus NS5A serine 225 phosphorylation revealed by super-resolution microscopy.

NS5A is a multi-functional phosphoprotein that plays a key role in hepatitis C virus (HCV) genome replication and assembly. The consequences of NS5A phosphorylation for HCV biology remain largely undefined. We previously identified serine 225 (S225) as a major phosphorylation site within the low complexity sequence 1 (LCSI) of NS5A and used a phosphoablatant mutant (S225A) to define the role of this phosphorylation event in genome replication, NS5A-host interactions and sub-cellular localisation. In this study, we investigate this further by raising an antiserum to S225 phosphorylated NS5A (pS225). Western blot analysis revealed that pS225 was predominantly in the hyper-phosphorylated NS5A species. Using a panel of phosphoablatant mutants of other phosphorylation sites in LCSI, we obtained evidence that is consistent with bidirectional hierarchical phosphorylation initiated by phosphorylation at S225. Using super-resolution microscopy (Airyscan and Expansion), we revealed a unique architecture of NS5A-positive punctae in HCV-infected cells; pS225 was present on the surface of these punctae, close to lipid droplets. Although S225 phosphorylation was not specifically affected by treatment with the NS5A-targeting direct acting antiviral agent daclatasvir, this resulted in the condensation of NS5A-positive punctae into larger structures, recapitulating the S225A phenotype. These data are consistent with a key role for S225 phosphorylation in the regulation of NS5A function.