Abstract
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 α (CKIα), polo-like kinase I (PlKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIα was studied previously and PlKI 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 γ and δ isoforms in the Huh7.5.1 cells. Both CaMKII γ and δ directly phosphorylated NS5A S235 in vitro. CaMKII γ or δ single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKIα plus CaMKII (γ or δ) 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 CKIα single knockdown. We conclude that CKIα-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII γ and δ may have negative roles in the HCV life cycle.
Highlights
Hepatitis C virus (HCV) is an enveloped virus with a positive single-stranded RNA genome
The HEK293T cells expressing NS3-5A without miR-122 expression were used for screening non-structural protein 5A (NS5A) kinases in order to avoid complication of the HCV life cycle that would alter the NS5A protein levels and interpretation of the drug effects on NS5A phosphorylation vs. viral life cycle
We exploited the NS3-5A expressing HEK293T kidney cells as a screening platform for kinases involved in NS5A serine 235 (S235) phosphorylation, a critical post-translational modification required for HCV replication [12, 14, 15]
Summary
Hepatitis C virus (HCV) is an enveloped virus with a positive single-stranded RNA genome. The viral genome encodes a polyprotein that is processed by the host and viral proteases into 3 structural (core, E1 and E2) and 7 non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins [1]. The structural proteins together with the host membranes make up the viral particles whereas the non-structural proteins are essential for a complete HCV life cycle. Many approved high efficiency drugs target the non-structural proteins for HCV infection that often leads to fibrosis, cirrhosis and cancer, if left unattended [2]. There are drugs that target the non-structural proteins with apparent enzymatic activities i.e. the NS3/4A protease complex and the RNA-dependent RNA polymerase NS5B [1, 3, 4].
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