title: Pim Kinase Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Entry.
abstract: The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼ 9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry.
  
  
  OBJECTIVE
  Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Chorong
authors.initials: C
authors.lastname: Park
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Saehong
authors.initials: S
authors.lastname: Min
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Eun-Mee
authors.initials: EM
authors.lastname: Park
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea.
authors.forename: Yun-Sook
authors.initials: YS
authors.lastname: Lim
authors.affiliation: Korea National Institute of Health, Cheongwon-gun, South Korea.
authors.forename: Sangmin
authors.initials: S
authors.lastname: Kang
authors.affiliation: Department of Infectious Diseases, Hamamatsu University School of Medicine, Hamamatsu, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, South Korea.
authors.forename: Eui-Cheol
authors.initials: EC
authors.lastname: Shin
authors.affiliation: National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea sbhwang@hallym.ac.kr.
authors.forename: Soon B
authors.initials: SB
authors.lastname: Hwang
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D055785:Gene Knockdown Techniques; D016174:Hepacivirus / Q000472:pathogenicity / Q000502:physiology*; D054884:Host-Pathogen Interactions / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D054730:Protein Interaction Domains and Motifs; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D051573:Proto-Oncogene Proteins c-pim-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000502:physiology*; D053586:Virus Internalization / Q000187:drug effects; D014779:Virus Replication / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D047428:Protein Kinase Inhibitors; D051573:Proto-Oncogene Proteins c-pim-1; D017361:Viral Nonstructural Proteins; C496634:PIM1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase