Knockdown of Autophagy Inhibits Infectious Hepatitis C Virus Release by the Exosomal Pathway

Abstract

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. We showed previously that HCV induces autophagy for viral persistence by preventing the innate immune response. Knockdown of autophagy reduces extracellular HCV release, although the precise mechanism remains unknown. In this study, we observed that knockdown of autophagy genes enhances intracellular HCV RNA and accumulates infectious virus particles in cells. Since HCV release is linked with the exosomal pathway, we examined whether autophagy proteins associate with exosomes in HCVinfected cells. We observed an association between HCV and the exosomal marker CD63 in autophagy knockdown cells. Subsequently, we observed that levels of extracellular infectious HCV were significantly lower in exosomes released from autophagy knockdown cells. To understand the mechanism for reduced extracellular infectious HCV in the exosome, we observed that an interferon (IFN)-stimulated BST-2 gene is upregulated in autophagy knockdown cells and associated with the exosome marker CD63, which may inhibit HCV assembly or release. Taken together, our results suggest a novel mechanism involving autophagy and exosome-mediated HCV release from infected hepatocytes. IMPORTANCEAutophagy plays an important role in HCV pathogenesis. Autophagy suppresses the innate immune response and promotes survival of virus-infected hepatocytes. The present study examined the role of autophagy in secretion of infectious HCV from hepatocytes. Autophagy promoted HCV trafficking from late endosomes to lysosomes, thus providing a link with the exosome. Inhibition of HCV-induced autophagy could be used as a strategy to block exosome-mediated virus transmission.

Exosome-Mediated Intercellular Communication between Hepatitis C Virus-Infected Hepatocytes and Hepatic Stellate Cells

“…HCV RNA can be found in exosomes, which can transmit HCV infection (7,10,11). We evaluated whether HCV RNA carried by HCV-exo can replicate in LX2 cells.…”

“…Exosomes isolated from liver endothelial cells stimulated by either type I or III IFNs can also suppress viral replication in HCV-infected liver cells (9). Recently, we have also shown the role of exosomes in HCV release from infected hepatocytes (10).…”

“…For infection, Huh7.5 or IHH (3 ϫ 10 5 cells) were incubated with HCV genotype 2a (clone JFH1) in a minimum volume of medium. After 8 h of virus adsorption on the hepatocytes, Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum was added as described previously (10). All the cells were maintained at 37°C in a humidified 5% CO 2 atmosphere.…”

The Autophagosomal SNARE Protein Syntaxin 17 Is an Essential Factor for the Hepatitis C Virus Life Cycle

“…However, induction of autophagy does not automatically mean that the process ends for all components in the autolysosome/lysosome. Syntaxin 17 might represent a trigger that enables a tight control of this process at a late point, affecting the number of viral particles that are finally released from the cell, i.e., in exosomes, or that are degraded in the lysosomal compartment (38).…”

“…It is established that HCV hijacks steps of the autophagic pathway (22,23,38). In light of the observation that the number of viral particles formed is much greater than the number of infectious viral particles that are finally released (16,17), regulation of the fusion between autophagosomes and lysosomes might fulfill a key function controlling the release of HCV.…”