title: 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 Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming 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 Δ1 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.
  
  
  OBJECTIVE
  While 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 Δ1 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.
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea.
authors.forename: Song-Hee
authors.initials: SH
authors.lastname: Han
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea.
authors.forename: Seong-Jun
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea.
authors.forename: Eun-Jung
authors.initials: EJ
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea.
authors.forename: Tae-Eun
authors.initials: TE
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea.
authors.forename: Jae-Su
authors.initials: JS
authors.lastname: Moon
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea.
authors.forename: Geon-Woo
authors.initials: GW
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea.
authors.forename: Seung-Hoon
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang, South Korea.
authors.forename: Kun
authors.initials: K
authors.lastname: Cho
authors.affiliation: Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang, South Korea.
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
authors.affiliation: Department of Pharmacy, CHA University, Pocheon-si, Gyeonggi-do, South Korea.
authors.forename: Woo Sung
authors.initials: WS
authors.lastname: Son
authors.affiliation: Western Seoul Center of Korea Basic Science Institute, Seoul, South Korea.
authors.forename: Jin-Kyu
authors.initials: JK
authors.lastname: Rhee
authors.affiliation: Department of Oral Microbiology and Immunology, DRI, and BK21 Program, School of Dentistry, Seoul National University, Seoul, South Korea.
authors.forename: Seung Hyun
authors.initials: SH
authors.lastname: Han
authors.affiliation: Department of Biotechnology, Yonsei University, Seodaemun-gu, Seoul, South Korea jwoh@yonsei.ac.kr.
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Oh
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
chemical_list: D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012694:Serine; D011493:Protein Kinase C; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase