The Roles of Alix and VPS4A in Autophagy and Endosomal Pathways and Their Relation to HBV Replication

Autophagic and endosomal pathways coordinately contribute to hepatitis B virus (HBV) production, with the endosomal sorting complex required for transport (ESCRT) components ALG-2-interacting protein X (Alix) and the vacuolar protein sorting 4A (VPS4A) playing important but mechanistically elusive roles. This study investigates the roles of Alix and VPS4A in HBV biogenesis within the context of endosomal trafficking and autophagy. Using gene silencing and overexpression of wild-type (WT) or dominant-negative (DN) mutants of Alix and VPS4A in HBV replication cell models, we found that Alix silencing increased intracellular HBV DNA and HBV surface antigen (HBsAg), extracellular HBsAg, and virions, while decreasing secreted naked capsids. It promoted HBsAg secretion along the early endosomes but reduced its transport to late endosomes and autophagosomes. Furthermore, Alix silencing impaired autophagosome formation by activating the AKT/MTOR pathway. In contrast, VPS4A silencing had minimal effects, whereas DN VPS4A significantly blocked HBV secretion by disrupting endosomal trafficking, promoting autophagosome formation and lysosome activity, ultimately leading to HBV degradation. Our findings demonstrate that the endosomal pathway is critical for HBV secretion when lysosomal activity is suppressed. Conversely, increased lysosomal function drives HBV degradation through the autophagosome-lysosome pathway.