Abstract
Hepatitis C virus (HCV) infection has been shown to induce autophagy but the mechanisms underpinning this process remain to be elucidated. Induction of autophagy requires the class III phosphatidylinositol 3-kinase, Vps34, which produces phosphatidylinositol 3-phosphate (PI3P) within the endoplasmic reticulum (ER) membrane. This recruits proteins with PI3P binding domains such as the double-FYVE-containing protein 1 (DFCP1). DFCP1 generates cup-shaped protrusions from the ER membrane, termed omegasomes, which provide a platform for the production of autophagosomes. Here we present data demonstrating that both Vps34 and DFCP1 are required for HCV genome replication, in the context of both a subgenomic replicon and virus infection, but did not affect virus entry or initial translation. Using live cell fluorescence microscopy we demonstrated that early during HCV infection the nascent viral genome replication complexes (identified by using non-structural protein NS5A as a marker) transiently colocalize with DFCP1-positive punctae (omegasomes), before the two structures move apart from each other. This observation is reminiscent of the transient association of LC3 and DFCP1 during omegasome formation, and therefore we propose that omegasomes are utilized by HCV to generate the double-membrane vesicles which are the hallmark of HCV replication complexes.
Highlights
Hepatitis C virus (HCV) is a positive-stranded RNA virus that establishes a chronic infection in 85% of infected individuals, leading to long-term liver disease such as cirrhosis and hepatocellular carcinoma
Wortmannin-treated cells showed a progressive loss of LC3-II over time when compared with the corresponding mock-treated controls, which is in agreement with inhibition of Vps34 class III phosphatidylinositol 3-kinase (PI3K) activity
In this study we demonstrated that early events in the generation of autophagosomes play a key role in the biogenesis of the membranous compartment required for HCV genome replication
Summary
Hepatitis C virus (HCV) is a positive-stranded RNA virus that establishes a chronic infection in 85% of infected individuals, leading to long-term liver disease such as cirrhosis and hepatocellular carcinoma. The recent development of an infectious cell culture system for HCV, based on the genotype 2a isolate, JFH-1 (Wakita et al, 2005), has allowed the detailed analysis of the molecular mechanisms of virus replication. In common with most positive strand RNA viruses, the entire life cycle of HCV occurs within the cytoplasm of infected cells. 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. 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
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