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.
Highlights
Hepatitis C virus (HCV) infection affects,170 million people worldwide and is a major cause of chronic liver disease including liver cirrhosis and hepatocellular carcinoma [1,2]
Colocalization was found between hepatitis C virus (HCV) proteins and mitochondria as revealed by staining with MitoTracker Red (Figure S1A) as well as between core, NS4B and NS5A and lipid droplets (LDs) that were detected by ADRP staining (Figure S1B) or with the lipid dye BODIPY (Table 1)
We note that analogous results were obtained when cells were analyzed already 24 hpi, at this time point HCV-specific signals were very much reduced as compared to 48 hpi
Summary
Hepatitis C virus (HCV) infection affects ,170 million people worldwide and is a major cause of chronic liver disease including liver cirrhosis and hepatocellular carcinoma [1,2]. The resulting polyprotein is co- and post-translationally processed by cellular and viral proteases into 10 different proteins that are required for RNA replication and virion formation. The N-terminal region of the polyprotein comprises the structural proteins core as well as envelope proteins 1 and 2 (E1 and E2) that build up the virus particle. C-terminal of E2 are p7 and nonstructural protein 2 (NS2) that are required for assembly of infectious HCV particles (reviewed in [6]). The latter is in addition a cysteine protease responsible for cleavage between NS2 and NS3 (reviewed in [4]).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
