A pivotal role of serine 225 phosphorylation in the function of hepatitis C virus NS5A revealed with the application of a phosphopeptide antiserum and super-resolution microscopy

NS5A is a multi-functional phosphoprotein that plays a key role in both viral replication and assembly. The identity of the kinases that phosphorylate NS5A, and the consequences for HCV biology, remain largely undefined. We previously identified serine 225 (S225) within low complexity sequence (LCS) I as a major phosphorylation site and used a phosphoablatant mutant (S225A) to define a role for S225 phosphorylation in the regulation of genome replication, interactions of NS5A with several host proteins and the sub-cellular localisation of NS5A. To investigate this further, we raised an antiserum to S225 phosphorylated NS5A (pS225). Western blot analysis revealed that pS225 was exclusively found in the hyper-phosphorylated NS5A species. Furthermore, using kinase inhibitors we demonstrated that S225 was phosphorylated by casein kinase 1α (CK1α) and polo-like kinase 1 (PLK1). Using a panel of phosphoablatant mutants of other phosphorylation sites in LCSI we obtained the first direct evidence of bidirectional hierarchical phosphorylation initiated by phosphorylation at S225.Using super-resolution microscopy (Airyscan and Expansion), we revealed a unique architecture of NS5A-positive clusters in HCV-infected cells - pS225 was concentrated on the surface of these clusters, close to lipid droplets. Pharmacological inhibition of S225 phosphorylation resulted in the condensation of NS5A-positive clusters into larger structures, recapitulating the S225A phenotype. Although S225 phosphorylation was not specifically affected by daclatasvir treatment, the latter also resulted in a similar condensation. These data are consistent with a key role for S225 phosphorylation in the regulation of NS5A function.ImportanceNS5A has obligatory roles in the hepatitis C virus lifecycle, and is proposed to be regulated by phosphorylation. As NS5A is a target for highly effective direct-acting antivirals (DAAs) such as daclatasvir (DCV) it is vital to understand how phosphorylation occurs and regulates NS5A function. We previously identified serine 225 (S225) as a major phosphorylation site. Here we used an antiserum specific for NS5A phosphorylated at S225 (pS225-NS5A) to identify which kinases phosphorylate this residue. Using super-resolution microscopy we showed that pS225 was present in foci on the surface of larger NS5A-positive clusters likely representing genome replication complexes. This location would enable pS225-NS5A to interact with cellular proteins and regulate the function and distribution of these complexes. Both loss of pS225 and DCV treatment resulted in similar changes to the structure of these complexes, suggesting that DAA treatment might target a function of NS5A that is also regulated by phosphorylation.