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

Journal article

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

Authors: Goonawardane, N; Yin, C; Roberts, GC; Zothner, C; Harris, M

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Scientific Reports
• Volume: 15
• Issue: 1
• Pages: 9567-9567
• Publication date: 2025-03-20
• DOI: 10.1038/s41598-025-93812-w
• EISSN: 2045-2322
• ISSN: 2045-2322
• Language: English
• Keywords: Hepatitis C virus; Phosphorylation cascade; NS5A; Protein phosphorylation; Cell biology; Biology; Phosphorylation; Serine; Virus; Hepacivirus; Virology