title: Temporal analysis of hepatitis C virus cell entry with occludin directed blocking antibodies.
abstract: Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America.
authors.forename: Marion
authors.initials: M
authors.lastname: Sourisseau
authors.forename: Maria L
authors.initials: ML
authors.lastname: Michta
authors.forename: Chati
authors.initials: C
authors.lastname: Zony
authors.forename: Benjamin
authors.initials: B
authors.lastname: Israelow
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Hopcraft
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Narbus
authors.forename: Ana
authors.initials: A
authors.lastname: Parra Martín
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Evans
mesh_terms: D019138:Antibodies, Blocking / Q000494:pharmacology*; D020868:Gene Silencing; D056945:Hep G2 Cells; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics / Q000472:pathogenicity; D054884:Host-Pathogen Interactions; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D009154:Mutation; D062793:Occludin / Q000235:genetics / Q000276:immunology*; D060225:Tetraspanin 28 / Q000378:metabolism; D019108:Tight Junctions; D013997:Time Factors; D014771:Virion / Q000472:pathogenicity / Q000502:physiology; D053586:Virus Internalization / Q000187:drug effects*; D014779:Virus Replication
chemical_list: D019138:Antibodies, Blocking; C554798:CD81 protein, human; C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D062793:Occludin; D060225:Tetraspanin 28
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