Abstract
Hepatitis C virus (HCV) induces autophagy to promote its replication, including its RNA replication, which can take place on double-membrane vesicles known as autophagosomes. However, how HCV induces the biogenesis of autophagosomes and how HCV RNA replication complex may be assembled on autophagosomes were largely unknown. During autophagy, crescent membrane structures known as phagophores first appear in the cytoplasm, which then progress to become autophagosomes. By conducting electron microscopy and in vitro membrane fusion assay, we found that phagophores induced by HCV underwent homotypic fusion to generate autophagosomes in a process dependent on the SNARE protein syntaxin 7 (STX7). Further analyses by live-cell imaging and fluorescence microscopy indicated that HCV-induced phagophores originated from the endoplasmic reticulum (ER). Interestingly, comparing with autophagy induced by nutrient starvation, the progression of phagophores to autophagosomes induced by HCV took significantly longer time, indicating fundamental differences in the biogenesis of autophagosomes induced by these two different stimuli. As the knockdown of STX7 to inhibit the formation of autophagosomes did not affect HCV RNA replication, and purified phagophores could mediate HCV RNA replication, the assembly of the HCV RNA replication complex on autophagosomes apparently took place during the formative stage of phagophores. These findings provided important information for understanding how HCV controlled and modified this important cellular pathway for its own replication.
Highlights
Autophagy is a catabolic process that is important for maintaining cellular homeostasis
Crescent membrane structures known as phagophores first appear in the cytoplasm, which expand to form enclosed double-membrane vesicles known as autophagosomes
We studied the biogenesis pathway of hepatitis C virus (HCV)-induced autophagosomes and demonstrated that phagophores induced by HCV originated from the endoplasmic reticulum and undergo homotypic fusion to generate autophagosomes, and that the HCV RNA replication complex is assembled on phagophores prior to the formation of autophagosomes
Summary
Autophagy is a catabolic process that is important for maintaining cellular homeostasis. It begins with the formation of membrane crescents termed phagophores or isolation membranes in the cytosol. In the canonical autophagic pathway, the class III phosphatidylinositol-3-kinase (PI3KC3) mediates the production of phosphatidylinositol-3-phosphate (PI3P), which recruits PI3P-binding proteins such as DFCP1 or WIPI to the PAS to form omegasomes [3]. This is followed by the recruitment of autophagy-related proteins ATG5 and ATG12, which are covalently linked, and ATG16, leading to the formation of phagophores. ATG5, ATG12 or ATG16 is often used as the marker for phagophores and LC3-II is often used as the marker for autophagosomes [6]
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