Abstract
Hepatitis C virus (HCV) infection is known to induce autophagosome accumulation as observed by the typical punctate cytoplasmic distribution of LC3B-II in infected cells. Previously, we showed that viral RNA-dependent RNA polymerase (NS5B) interacts with ATG5, a major component of the autophagy elongation complex that is involved in the formation of double-membrane vesicles (DMV), and demonstrated that the autophagy elongation complex (ATG5-12/16L1) but not LC3B is required for proper membranous web formation. In this study, the colocalization and in situ interaction of all HCV replicase components with the constituent of the autophagy elongation complex and LC3B were analyzed. The results clearly show the recruitment of the elongation complex to the site of viral replication. Using in situ proximity ligation assay, we show that ATG5, but not ATG16L1, interacts with several HCV replicase components suggesting that the recruitment is directed via the ATG5-12 conjugate. Interestingly, no E3-like conjugation activity of ATG5-12/16L1 can be detected at the at HCV replication site since LC3B-II is not found along with the elongation complex at the site of viral replication. In agreement with this result, no sign of in situ interaction of LC3B with the replicase components is observed. Finally, using dominant negative forms of ATG proteins, we demonstrate that ATG5-12 conjugate, but not LC3-II formation, is critical for viral replication. Altogether, these findings suggest that although HCV needs the elongation complex for its replication, it has developed a mechanism to avoid canonical LC3-II accumulation at viral replication site.
Highlights
Hepatitis C virus (HCV) infection leads to a wide spectrum of diseases ranging from asymptomatic to end-stage liver diseases including cirrhosis and hepatocellular carcinoma [1]
Autophagy elongation complex but not LC3 is involved in HCV replication viral NS3, NS4A, NS5A, and NS5B in merged image panels are shown
We showed that HCV RNA-dependent RNA polymerase (RdRp) colocalizes and interacts with ATG5, a component of the elongation complex [28]
Summary
Hepatitis C virus (HCV) infection leads to a wide spectrum of diseases ranging from asymptomatic to end-stage liver diseases including cirrhosis and hepatocellular carcinoma [1]. HCV is an enveloped, positive-strand RNA virus that belongs to the Flaviviridae family. Autophagy elongation complex but not LC3 is involved in HCV replication coding regions (NCRs). HCV replication is marked by the formation of a membrane-associated replication complex with a unique membrane alteration referred to as the membranous web [3]. The majority of the membranous structures found within the HCV replication site are composed of double membrane vesicles (DMVs) suggesting that autophagy is involved in the establishment of the HCV replication scaffold [4,5,6]. DMVs are strongly suspected of being the primary site of HCV replicase localization where active viral RNA replication occurs [4, 7, 8]
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