title: Persistent hepatitis C virus infection impairs ribavirin antiviral activity through clathrin-mediated trafficking of equilibrative nucleoside transporter 1.
abstract: Ribavirin (RBV) continues to be an important component of interferon-free hepatitis C treatment regimens, as RBV alone does not inhibit hepatitis C virus (HCV) replication effectively; the reason for this ineffectiveness has not been established. In this study, we investigated the RBV resistance mechanism using a persistently HCV-infected cell culture system. The antiviral activity of RBV against HCV was progressively impaired in the persistently infected culture, whereas interferon lambda 1 (IFN-λ1), a type III IFN, showed a strong antiviral response and induced viral clearance. We found that HCV replication in persistently infected cultures induces an autophagy response that impairs RBV uptake by preventing the expression of equilibrative nucleoside transporter 1 (ENT1). The Huh-7.5 cell line treated with an autophagy inducer, Torin 1, downregulated membrane expression of ENT1 and terminated RBV uptake. In contrast, the autophagy inhibitors hydroxychloroquine (HCQ), 3-methyladenine (3-MA), and bafilomycin A1 (BafA1) prevented ENT1 degradation and enhanced RBV antiviral activity. The HCV-induced autophagy response, as well as treatment with Torin 1, degrades clathrin heavy chain expression in a hepatoma cell line. Reduced expression of the clathrin heavy chain by HCV prevents ENT1 recycling to the plasma membrane and forces ENT1 to the lysosome for degradation. This study provides a potential mechanism for the impairment of RBV antiviral activity in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy for improving RBV antiviral activity against HCV infection.
  
  
  OBJECTIVE
  The results from this work will allow a review of the competing theories of antiviral therapy development in the field of HCV virology. Ribavirin (RBV) remains an important component of interferon-free hepatitis C treatment regimens. The reason why RBV alone does not inhibit HCV replication effectively has not been established. This study provides a potential mechanism for why RBV antiviral activity is impaired in persistently HCV-infected cell cultures and suggests that inhibition of the HCV-induced autophagy response could be used as a strategy to increase RBV antiviral activity against HCV infection. Therefore, it is anticipated that this work would generate a great deal of interest, not only among virologists but also among the general public.
authors.affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Panigrahi
authors.affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Partha K
authors.initials: PK
authors.lastname: Chandra
authors.affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Pauline
authors.initials: P
authors.lastname: Ferraris
authors.affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA Department of Medicine, Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Ramazan
authors.initials: R
authors.lastname: Kurt
authors.affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Kyoungsub
authors.initials: K
authors.lastname: Song
authors.affiliation: Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Garry
authors.affiliation: Neurological Cancer Research, Stanley S. Scott Cancer Center, New Orleans, Louisiana, USA.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Reiss
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON, Canada.
authors.forename: Imogen R
authors.initials: IR
authors.lastname: Coe
authors.affiliation: Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Science, Chiba University, Chiba, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Furihata
authors.affiliation: Department of Medicine, Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Balart
authors.affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Tong
authors.initials: T
authors.lastname: Wu
authors.affiliation: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA Department of Medicine, Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, USA sdash@tulane.edu.
authors.forename: Srikanta
authors.initials: S
authors.lastname: Dash
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism*; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D004351:Drug Resistance; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism*; D016174:Hepacivirus / Q000187:drug effects*; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D021381:Protein Transport; D012254:Ribavirin / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D002966:Clathrin; D033721:Equilibrative Nucleoside Transporter 1; D012254:Ribavirin