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.

Early events in the generation of autophagosomes are required for the formation of membrane structures involved in hepatitis C virus genome replication

“…Of note, the timescale of this process as observed in this study (up to 10 min from formation of the NS5A-DFCP1 puncta to their eventual dissociation -see Figs 6 and 7), is consistent with the timescale observed previously for the transient association of GFP-DFCP1 and RFP-LC3 during autophagosome formation (also approximately 10 min) (Axe et al, 2008). The difference in size between autophagosomes (v1.5 mm) and DMVs (100-150 nm) (Romero-Brey et al, 2012) is also consistent with the hypothesis that DMVs derive from autophagosomes.…”

“…Our data complement the recent, elegant study of Romero-Brey et al (2012), who showed that the membranous web in HCV-infected cells comprised predominantly DMVs and derived from the ER. In particular, they identified DMVs as protrusions from the ER membrane.…”

“…The potential parallels between this process and the formation of autophagosomes are striking, particularly with regard to the role of DFCP1 in mediating the formation of omegasomes at ER membranes (Axe et al, 2008). Romero-Brey et al (2012) also highlighted the similarities between the morphology of the membrane rearrangements seen in HCV infection and those of unrelated viruses such as the coronaviruses and arteriviruses.…”

“…NS4B and NS5A were the most likely candidates as both have been shown to individually generate DMV or single-membrane vesicles (SMV) (Paul et al, 2013;Romero-Brey et al, 2012). Additionally, recent evidence has pointed to a role for NS4B in the induction of autophagy and accumulation of autophagosomes (Su et al, 2011;Wang et al, 2015).…”

“…The virus induces rearrangements of cytoplasmic membranes to form a structure called the 'membranous web' (Egger et al, 2002;Gosert et al, 2003) -a cluster of mainly double-membraned vesicles (DMV) that constitutes a replication factory where genome replication takes place. Despite recent advances in understanding the architecture of the membranous web (Paul et al, 2013;Romero-Brey et al, 2012), the biogenesis of this structure remains unclear. Although it is thought to derive from the endoplasmic reticulum (ER), the mechanisms by which DMV are produced from ER membranes, and the cellular factors involved in this process, have not been identified.…”