Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases

Phosphorylation at serine 235 (S235) of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays a critical role in the viral life cycle. For medical and virological interests, we exploited the HEK293T kidney cells to test 3 candidate protein kinases on NS5A S235 phosphorylation. Inhibitors that inhibit casein kinase I alpha (CKI alpha), polo-like kinase I (PIKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIa was studied previously and PIKI had severe cytotoxicity, thus CaMKII was selected for validation in the Huh7.5.1 liver cells. In the HCV (J6/JFH1)-infected Huh7.5.1 cells, CaMKII inhibitor reduced NS5A S235 phosphorylation and HCV RNA levels without apparent cytotoxicity. RT-PCR analysis showed expression of CaMKII gamma and delta isoforms in the Huh7.5.1 cells. Both CaMKII gamma and delta directly phosphorylated NS5A S235 in vitro. CaMKII gamma or delta single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKI alpha plus CaMKII (gamma or delta) double knockdown reduced NS5A S235 phosphorylation and reduced HCV RNA levels; however, the HCV RNA levels were higher than those in the infected cells with CKI alpha single knockdown. We conclude that CKI alpha-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII gamma and delta may have negative roles in the HCV life cycle.