Characterization of hepatitis C virus NS3 modifications in the context of replication.

Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV non-structural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.