title: Early events in the generation of autophagosomes are required for the formation of membrane structures involved in hepatitis C virus genome replication.
abstract: Hepatitis C virus (HCV) infection has been shown to induce autophagy but the mechanisms underpinning this process remain to be elucidated. Induction of autophagy requires the class III phosphatidylinositol 3-kinase, Vps34, which produces phosphatidylinositol 3-phosphate (PI3P) within the endoplasmic reticulum (ER) membrane. This recruits proteins with PI3P binding domains such as the double-FYVE-containing protein 1 (DFCP1). DFCP1 generates cup-shaped protrusions from the ER membrane, termed omegasomes, which provide a platform for the production of autophagosomes. Here we present data demonstrating that both Vps34 and DFCP1 are required for HCV genome replication, in the context of both a subgenomic replicon and virus infection, but did not affect virus entry or initial translation. Using live cell fluorescence microscopy we demonstrated that early during HCV infection the nascent viral genome replication complexes (identified by using non-structural protein NS5A as a marker) transiently colocalize with DFCP1-positive punctae (omegasomes), before the two structures move apart from each other. This observation is reminiscent of the transient association of LC3 and DFCP1 during omegasome formation, and therefore we propose that omegasomes are utilized by HCV to generate the double-membrane vesicles which are the hallmark of HCV replication complexes.
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Bjorn-Patrick
authors.initials: BP
authors.lastname: Mohl
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bartlett
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Jamel
authors.initials: J
authors.lastname: Mankouri
authors.affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Harris
mesh_terms: D001343:Autophagy; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D058546:Class III Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000821:virology; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006526:Hepatitis C / Q000503:physiopathology* / Q000821:virology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000821:virology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D014779:Virus Replication
chemical_list: D002352:Carrier Proteins; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; C426003:ZFYVE1 protein, human; D058546:Class III Phosphatidylinositol 3-Kinases