title: Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication.
abstract: OBJECTIVE
  Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives.
  
  
  METHODS
  We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H.
  
  
  RESULTS
  NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web.
  
  
  CONCLUSIONS
  Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis-a new paradigm in antiviral therapy.
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Carola
authors.initials: C
authors.lastname: Berger
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Inés
authors.initials: I
authors.lastname: Romero-Brey
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Danijela
authors.initials: D
authors.lastname: Radujkovic
authors.affiliation: CNRS, UMR5086, Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, Lyon, France; Labex Ecofect (ANR-11-LABX-0042), University of Lyon, Lyon, France; Faculté de Pharmacie (ISPB), Lyon, France.
authors.forename: Raphael
authors.initials: R
authors.lastname: Terreux
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Margarita
authors.initials: M
authors.lastname: Zayas
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Paul
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Harak
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Hoppe
authors.affiliation: Bristol-Myers Squibb Research and Development, Wallingford, Connecticut.
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.affiliation: CNRS, UMR5086, Bases Moléculaires et Structurales des Systèmes Infectieux, Institut de Biologie et Chimie des Protéines, Lyon, France; Labex Ecofect (ANR-11-LABX-0042), University of Lyon, Lyon, France.
authors.forename: Francois
authors.initials: F
authors.lastname: Penin
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Lohmann
authors.affiliation: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; German Centre for Infection Research, Heidelberg University, Heidelberg, Germany. Electronic address: Ralf_Bartenschlager@med.uni-heidelberg.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D002219:Carbamates; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure / Q000821:virology; D015195:Drug Design; D024882:Drug Resistance, Viral; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology*; D015778:Minor Histocompatibility Antigens; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation; D055503:Protein Multimerization; D011759:Pyrrolidines; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D007093:Imidazoles; D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor); D011480:Protease Inhibitors; D011759:Pyrrolidines; D014633:Valine; D017361:Viral Nonstructural Proteins; C402182:phosphatidylinositol phosphate 4-kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir
keywords: Chronic Hepatitis C; Drug Mechanism; Viral Replication; Viral Replication Factory