title: Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication.
abstract: The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.
authors: MRC Virology Unit, Church Street, Glasgow G11 5JR, United Kingdom. p.targett-adams@mrcvu.gla.ac.uk
authors: Paul
authors: P
authors: Targett-Adams
authors: Steeve
authors: S
authors: Boulant
authors: John
authors: J
authors: McLauchlan
mesh_terms: D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D012330:RNA, Double-Stranded / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral