Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

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 th...

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Bibliographic Details
Published in:	PLoS pathogens 2012-12, Vol.8 (12), p.e1003056-e1003056
Main Authors:	Romero-Brey, Inés, Merz, Andreas, Chiramel, Abhilash, Lee, Ji-Young, Chlanda, Petr, Haselman, Uta, Santarella-Mellwig, Rachel, Habermann, Anja, Hoppe, Simone, Kallis, Stephanie, Walther, Paul, Antony, Claude, Krijnse-Locker, Jacomine, Bartenschlager, Ralf
Format:	Article
Language:	eng
Subjects:	Architecture Biology Biosynthesis Cell culture Cell Line Colleges & universities Drug resistance Endoplasmic Reticulum - metabolism Endoplasmic Reticulum - ultrastructure Endoplasmic Reticulum - virology Genetic aspects Genomes Health aspects Hepacivirus - physiology Hepacivirus - ultrastructure Hepatitis Hepatitis C - metabolism Hepatitis C - pathology Hepatitis C virus Humans Infections Intracellular Membranes - metabolism Intracellular Membranes - ultrastructure Intracellular Membranes - virology Liver cirrhosis Liver diseases Medicine Microscopy Microscopy, Electron, Transmission - methods Physiological aspects Plasmids Proteins Reading RNA RNA, Viral - biosynthesis Viral genetics Virus Replication - physiology Viruses
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Summary:	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.
ISSN:	1553-7374 1553-7366 1553-7374