title: Phosphatidylinositol 4,5-bisphosphate is an HCV NS5A ligand and mediates replication of the viral genome.
abstract: OBJECTIVE
  Phosphoinositides (PIs) bind and regulate localization of proteins via a variety of structural motifs. PI 4,5-bisphosphate (PI[4,5]P2) interacts with and modulates the function of several proteins involved in intracellular vesicular membrane trafficking. We investigated interactions between PI(4,5)P2 and hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and effects on the viral life cycle.
  
  
  METHODS
  We used a combination of quartz crystal microbalance, circular dichroism, molecular genetics, and immunofluorescence to study specific binding of PI(4,5)P2 by the HCV NS5A protein. We evaluated the effects of PI(4,5)P2 on the function of NS5A by expressing wild-type or mutant forms of Bart79I or FL-J6/JFH-5'C19Rluc2AUbi21 RNA in Huh7 cells. We also studied the effects of strategies designed to inhibit PI(4,5)P2 on HCV replication in these cells.
  
  
  RESULTS
  The N-terminal amphipathic helix of NS5A bound specifically to PI(4,5)P2, inducing a conformational change that stabilized the interaction between NS5A and TBC1D20, which is required for HCV replication. A pair of positively charged residues within the amphipathic helix (the basic amino acid PI(4,5)P2 pincer domain) was required for PI(4,5)P2 binding and replication of the HCV-RNA genome. A similar motif was found to be conserved across all HCV isolates, as well as amphipathic helices of many pathogens and apolipoproteins.
  
  
  CONCLUSIONS
  PI(4,5)P2 binds to HCV NS5A to promote replication of the viral RNA genome in hepatocytes. Strategies to disrupt this interaction might be developed to inhibit replication of HCV and other viruses.
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California.
authors.forename: Nam-Joon
authors.initials: NJ
authors.lastname: Cho
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California.
authors.forename: Choongho
authors.initials: C
authors.lastname: Lee
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Division of Infectious Diseases, Stanford University School of Medicine, Stanford, California.
authors.forename: Phillip S
authors.initials: PS
authors.lastname: Pang
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Division of Infectious Diseases, Stanford University School of Medicine, Stanford, California.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Pham
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Fram
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California.
authors.forename: Khanh
authors.initials: K
authors.lastname: Nguyen
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California.
authors.forename: Anming
authors.initials: A
authors.lastname: Xiong
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California.
authors.forename: Ella H
authors.initials: EH
authors.lastname: Sklan
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California.
authors.forename: Menashe
authors.initials: M
authors.lastname: Elazar
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California.
authors.forename: Elif S
authors.initials: ES
authors.lastname: Koytak
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California.
authors.forename: Caroline
authors.initials: C
authors.lastname: Kersten
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California.
authors.forename: Kay K
authors.initials: KK
authors.lastname: Kanazawa
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California.
authors.forename: Curtis W
authors.initials: CW
authors.lastname: Frank
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, California; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California; Veterans Administration Medical Center, Palo Alto, California. Electronic address: jeffrey.glenn@stanford.edu.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Glenn
mesh_terms: D002470:Cell Survival; D002942:Circular Dichroism; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D022781:Hepatocytes; D006801:Humans; D008856:Microscopy, Fluorescence; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D017433:Protein Structure, Secondary; D058525:Quartz Crystal Microbalance Techniques; D017423:Sequence Analysis, RNA; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication; D020761:rab1 GTP-Binding Proteins / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D017361:Viral Nonstructural Proteins; D020761:rab1 GTP-Binding Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: Antiviral Strategies; Phospholipid; QCM; Signaling Molecule