title: Chaperone-Mediated Autophagy Targets IFNAR1 for Lysosomal Degradation in Free Fatty Acid Treated HCV Cell Culture.
abstract: BACKGROUND
  Hepatic steatosis is a risk factor for both liver disease progression and an impaired response to interferon alpha (IFN-α)-based combination therapy in chronic hepatitis C virus (HCV) infection. Previously, we reported that free fatty acid (FFA)-treated HCV cell culture induces hepatocellular steatosis and impairs the expression of interferon alpha receptor-1 (IFNAR1), which is why the antiviral activity of IFN-α against HCV is impaired.
  
  
  OBJECTIVE
  To investigate the molecular mechanism by which IFNAR1 expression is impaired in HCV cell culture with or without free fatty acid-treatment.
  
  
  METHODS
  HCV-infected Huh 7.5 cells were cultured with or without a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in HCV-infected culture was visualized by oil red staining. Clearance of HCV in FFA cell culture treated with type I IFN (IFN-α) and Type III IFN (IFN-λ) was determined by Renilla luciferase activity, and the expression of HCV core was determined by immunostaining. Activation of Jak-Stat signaling in the FFA-treated HCV culture by IFN-α alone and IFN-λ alone was examined by Western blot analysis and confocal microscopy. Lysosomal degradation of IFNAR1 by chaperone-mediated autophagy (CMA) in the FFA-treated HCV cell culture model was investigated.
  
  
  RESULTS
  FFA treatment induced dose-dependent hepatocellular steatosis and lipid droplet accumulation in HCV-infected Huh-7.5 cells. FFA treatment of infected culture increased HCV replication in a concentration-dependent manner. Intracellular lipid accumulation led to reduced Stat phosphorylation and nuclear translocation, causing an impaired IFN-α antiviral response and HCV clearance. Type III IFN (IFN-λ), which binds to a separate receptor, induces Stat phosphorylation, and nuclear translocation as well as antiviral clearance in FFA-treated HCV cell culture. We show here that the HCV-induced autophagy response is increased in FFA-treated cell culture. Pharmacological inhibitors of lysosomal degradation, such as ammonium chloride and bafilomycin, prevented IFNAR1 degradation in FFA-treated HCV cell culture. Activators of chaperone-mediated autophagy, including 6-aminonicotinamide and nutrient starvation, decreased IFNAR1 levels in Huh-7.5 cells. Co-immunoprecipitation, colocalization and siRNA knockdown experiments revealed that IFNAR1 but not IFNLR1 interacts with HSC70 and LAMP2A, which are core components of chaperone-mediated autophagy (CMA).
  
  
  CONCLUSIONS
  Our study presents evidence indicating that chaperone-mediated autophagy targets IFNAR1 degradation in the lysosome in FFA-treated HCV cell culture. These results provide a mechanism for why HCV induced autophagy response selectively degrades type I but not the type III IFNAR1.
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Ramazan
authors.initials: R
authors.lastname: Kurt
authors.affiliation: Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Partha K
authors.initials: PK
authors.lastname: Chandra
authors.affiliation: Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Fatma
authors.initials: F
authors.lastname: Aboulnasr
authors.affiliation: Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Panigrahi
authors.affiliation: Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Pauline
authors.initials: P
authors.lastname: Ferraris
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Yucel
authors.initials: Y
authors.lastname: Aydin
authors.affiliation: Neurological Cancer Research, Stanley S Scott Cancer Center, LSU Health Science Center, New Orleans, Louisiana, United States of America.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Reiss
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Tong
authors.initials: T
authors.lastname: Wu
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Balart
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, Tulane University School of Medicine, New Orleans, Louisiana, United States of America; Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
authors.forename: Srikanta
authors.initials: S
authors.lastname: Dash
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D002460:Cell Line; D002478:Cells, Cultured; D005230:Fatty Acids, Nonesterified / Q000378:metabolism* / Q000494:pharmacology; D005234:Fatty Liver / Q000235:genetics / Q000378:metabolism / Q000821:virology; D015870:Gene Expression; D016174:Hepacivirus / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007370:Interferon Type I / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D053612:Janus Kinases / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D059748:Proteolysis / Q000187:drug effects; D053633:Receptor, Interferon alpha-beta / Q000235:genetics / Q000378:metabolism*; D050791:STAT Transcription Factors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D005230:Fatty Acids, Nonesterified; C507964:IFNAR1 protein, human; D007370:Interferon Type I; D050791:STAT Transcription Factors; D053633:Receptor, Interferon alpha-beta; D007371:Interferon-gamma; D053612:Janus Kinases