title: Identification of GBF1 as a cellular factor required for hepatitis C virus RNA replication.
abstract: In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.
authors.affiliation: CNRS-UMR8161, Institut de Biologie de Lille, 1 rue du Professeur Calmette, BP447, 59021 Lille cedex, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Goueslain
authors.forename: Khaled
authors.initials: K
authors.lastname: Alsaleh
authors.forename: Pauline
authors.initials: P
authors.lastname: Horellou
authors.forename: Philippe
authors.initials: P
authors.lastname: Roingeard
authors.forename: Véronique
authors.initials: V
authors.lastname: Descamps
authors.forename: Gilles
authors.initials: G
authors.lastname: Duverlie
authors.forename: Yann
authors.initials: Y
authors.lastname: Ciczora
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Wychowski
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
authors.forename: Yves
authors.initials: Y
authors.lastname: Rouillé
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D020662:Guanine Nucleotide Exchange Factors / Q000187:drug effects / Q000378:metabolism*; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008854:Microscopy, Electron; D012367:RNA, Viral / Q000096:biosynthesis*; D014779:Virus Replication / Q000187:drug effects
chemical_list: C120445:GBF1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D012367:RNA, Viral; D020126:Brefeldin A; D020727:ADP-Ribosylation Factors