title: Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.
abstract: Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly.
  
  
  
  Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy.
  
  
  
  Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes.
  
  
  
  In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection.
authors.affiliation: CIRI - International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, F-69007, Lyon, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Boson
authors.affiliation: CIRI - International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, F-69007, Lyon, France.
authors.forename: Solène
authors.initials: S
authors.lastname: Denolly
authors.affiliation: CIRI - International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, F-69007, Lyon, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Turlure
authors.affiliation: Plateau Technique Imagerie/Microcopie, Lyon Bio Image, SFR-BioSciences, ENS de Lyon, Inserm US8, CNRS UMS3444, UCBL, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Chamot
authors.affiliation: CIRI - International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, F-69007, Lyon, France.
authors.forename: Marlène
authors.initials: M
authors.lastname: Dreux
authors.affiliation: CIRI - International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, F-69007, Lyon, France. Electronic address: flcosset@ens-lyon.fr.
authors.forename: François-Loïc
authors.initials: FL
authors.lastname: Cosset
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002219:Carbamates; D045744:Cell Line, Tumor; D016679:Genome, Viral; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D011759:Pyrrolidines; D034443:RNA Transport / Q000187:drug effects*; D012367:RNA, Viral / Q000378:metabolism*; D014633:Valine / Q000031:analogs & derivatives; D014758:Viral Core Proteins / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D007093:Imidazoles; D011759:Pyrrolidines; D012367:RNA, Viral; D014633:Valine; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; C080107:NS4B protein, flavivirus; C091015:glycoprotein E2, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir
keywords: Chronic Hepatitis C; DAA; Direct-Acting Antiviral Agent