title: Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides.
abstract: Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies is HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation.
authors.affiliation: Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Vineela
authors.initials: V
authors.lastname: Chukkapalli
authors.affiliation: Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Kristi L
authors.initials: KL
authors.lastname: Berger
authors.affiliation: Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Kelly
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Meryl
authors.initials: M
authors.lastname: Thomas
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Deiters
authors.affiliation: Department of Microbiology, The University of Chicago, Chicago, IL 60637, USA. Electronic address: grandall@bsd.uchicago.edu.
authors.forename: Glenn
authors.initials: G
authors.lastname: Randall
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002219:Carbamates; D002460:Cell Line; D016174:Hepacivirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D015778:Minor Histocompatibility Antigens; D010716:Phosphatidylinositols / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011759:Pyrrolidines; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D007093:Imidazoles; D015778:Minor Histocompatibility Antigens; D010716:Phosphatidylinositols; D011759:Pyrrolidines; D017361:Viral Nonstructural Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D014633:Valine; C549273:daclatasvir
keywords: Direct-acting anti-virals; HCV replication complex; Phosphatidylinositol-4-kinase alpha; Phosphatidylinositol-4-phosphate