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. All positive-strand RNA viruses replicate in the cytoplasm in distinct membranous compartments acting as ‘replication factories’. Membranes building up these factories are recruited from different sources and serve as platforms for the assembly of multi-subunit protein complexes (the ‘replicase’) that catalyze the amplification of the viral RNA genome. In this study we found that hepatitis C virus (HCV), a major causative agent of chronic liver disease, induces profound remodeling of primarily endoplasmic reticulum-derived membranes. Surprisingly, the 3D architecture of these membrane rearrangements is similar to those induced by the unrelated picorna- and coronaviruses, but in striking contrast to the closely related flaviviruses. Early in infection HCV induces double membrane vesicles (DMVs) that emerge as protrusions of the ER; later on, HCV induces in addition multi-membrane vesicles that are probably the result of a cellular stress reaction and that are reminiscent to an autophagic response. These profound membrane rearrangements are induced by the concerted action of HCV-encoded nonstructural proteins of which NS5A is the only one capable to induce DMVs. These results provide important insights into the 3D architecture of the membrane alterations induced by HCV and reveal unexpected similarities between HCV and the very distantly related picorna- and coronaviruses.

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

Background & Aims Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. Methods We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. Results NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. Conclusions Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis—a new paradigm in antiviral therapy.