Ultrastructural and biochemical analyses of hepatitis C virus-associated host cell membranes

Like most other positive-strand RNA viruses, hepatitis C virus (HCV) induces changes in the host cell's membranes, resulting in a membranous web. The non-structural proteins of the viral replication complex are thought to be associated with these newly synthesized membranes. We studied this phenomenon, using a Huh7.5 cell clone displaying high levels of replication of a subgenomic replicon of the JFH-1 strain. Electron microscopy of ultrathin sections of these cells revealed the presence of numerous double membrane vesicles (DMVs), resembling those observed for other RNA viruses such as poliovirus and coronavirus. Some sections had more discrete multivesicular units consisting of circular concentric membranes organized into clusters surrounded by a wrapping membrane. These structures were highly specific to HCV as they were not detected in naive Huh7.5 cells. Preparations enriched in these structures were separated from other endoplasmic reticulum-derived membranes by cell cytoplasm homogenization and ultracentrifugation on a sucrose gradient. They were found to contain the non-structural NS3 and NS5A HCV proteins, HCV RNA and LC3-II, a specific marker of autophagic membranes. By analogy to other viral models, HCV may induce DMVs by activating the autophagy pathway. This could represent a strategy to conceal the viral RNA and help the virus to evade double-stranded RNA-triggered host antiviral responses. More detailed characterization of these virus-cell interactions may facilitate the development of new treatments active against HCV and other RNA viruses that are dependent on newly synthesized cellular membranes for replication.