Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

Abstract

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the ⌬1 finger loop region that interacts with the thumb subdomain of NS5B. Colonyforming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its ⌬1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCEWhile the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the ⌬1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

Hepatitis C Virus RNA-Dependent RNA Polymerase Interacts with the Akt/PKB Kinase and Induces Its Subcellular Relocalization

“…2). RNA synthesis defects in HCV NS5B mutants that mimic phosphorylation at Ser residues have been previously reported (35). In any case, further experiments with proteins carrying different combinations of mutated residues as well as with deletion mutants should be performed to precisely map the residues that are phosphorylated by Akt/PKB and its subsequent effects on polymerase activity and subcellular localization.…”

Phosphorylation at the N-terminal finger subdomain of a viral RNA-dependent RNA polymerase

“…Recently, two serine residues, Ser29 and Ser42, have been identified as phosphorylation sites on HCV NS5B (from the viral genotype 1b), indicating that PRK2 regulates this viral enzyme via phosphorylation [13]. In the current report, we expand this research on the regulation of the finger subdomain of HCV NS5B genotype 2a, via phosphorylation.…”

“…These data are strongly consistent with our previous functional results, and as Ser76 is conserved across HCV genotypes, these data confirm the importance of Ser29 and Ser76 for both viral genotypes as phosphorylation sites. Importantly, since Han et al [13] finely mapped the phosphorylation sites of recombinant PRK2 only at the region covering residues 28 and 53 of NS5B, our in vitro kinase results utilizing a crude cytosolic extract would imply that NS5B Ser76 is phosphorylated by a cellular kinase different from PRK2.…”

“…3B). Interestingly, this residue was also recently reported to be phosphorylated in vitro by PRK2 when using HCV NS5B from genotype 1b [13]. The second peptide identified in the spectrum had precursors charged at 491.7517 m/z, which indicated that Ser76 was phosphorylated (Fig.…”

“…In light of the knowledge regarding phosphorylation of the Nterminal fingers subdomain of NS5B protein from genotype 1b [12,13], we decided to analyze the regulation of the phosphorylation of NS5B from genotype 2a, looking for conserved potential residues. For this, we collected sequences of NS5B proteins from the main viral genotypes, and we performed a multiple sequence alignment with the first 188 amino acid from the N-terminus of the viral polymerase ( Supplementary Fig.…”