title: Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.
abstract: All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.
authors.affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.
authors.forename: Inés
authors.initials: I
authors.lastname: Romero-Brey
authors.forename: Andreas
authors.initials: A
authors.lastname: Merz
authors.forename: Abhilash
authors.initials: A
authors.lastname: Chiramel
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Lee
authors.forename: Petr
authors.initials: P
authors.lastname: Chlanda
authors.forename: Uta
authors.initials: U
authors.lastname: Haselman
authors.forename: Rachel
authors.initials: R
authors.lastname: Santarella-Mellwig
authors.forename: Anja
authors.initials: A
authors.lastname: Habermann
authors.forename: Simone
authors.initials: S
authors.lastname: Hoppe
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kallis
authors.forename: Paul
authors.initials: P
authors.lastname: Walther
authors.forename: Claude
authors.initials: C
authors.lastname: Antony
authors.forename: Jacomine
authors.initials: J
authors.lastname: Krijnse-Locker
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D016174:Hepacivirus / Q000502:physiology / Q000648:ultrastructure; D006526:Hepatitis C / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology; D046529:Microscopy, Electron, Transmission / Q000379:methods; D012367:RNA, Viral / Q000096:biosynthesis*; D014779:Virus Replication / Q000502:physiology
chemical_list: D012367:RNA, Viral