INSERM U966, Université Franç ois Rabelais and CHRU de Tours, FranceLike 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. INTRODUCTIONThe formation of a membrane-associated replication complex, consisting of viral proteins, replicating RNA, altered cellular membranes and other host factors, is a hallmark of all positive-strand RNA viruses including those infecting mammalian, insect or plant cells (Miller & Krijnse-Locker, 2008;Mackenzie, 2005). Depending on the virus, replication may occur on rearranged convoluted membranes, single or double membrane vesicles of various sizes derived from the endoplasmic reticulum (ER), the intermediate compartment between the ER and the Golgi apparatus, the trans-Golgi network, mitochondria or even lysosomes (Miller & KrijnseLocker, 2008). These membrane structures induced by positive-strand RNA viruses probably serve as a scaffold for the assembly of viral replication complexes by providing an organization and environment facilitating viral replication (Lyle et al., 2002; Schwartz et al., 2002). Some positivestranded RNA viruses including some strains of poliovirus (Jackson et al., 2005), coxsackievirus B3 (Wong et al., 2008), dengue virus (Lee et al., 2008) and mouse hepatitis coronavirus (Prentice et al., 2004) may take over the host autophagy machinery to facilitate their own replication. These positive-stranded RNA viruses trigger autophagosome-like formation without triggering the ultimate degradati...

Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.