Background & Aims: Autophagic and endosomal pathways coordinately contribute to hepatitis B virus (HBV) virions and subviral particles (SVPs) production. To date, limited evidence supports that HBV and exosomes have a common pathway for their biogenesis and secretion. The final steps of HBV production and release have not yet been well studied.

Methods & Results: In the present study, we examined the production and release of HBV virions and SVPs by using GW4869, a small molecule inhibiting ceramide-mediated inward membrane budding. Neutral sphingomyelinase (nSMase), the target of GW4869, was silenced to confirm the results obtained. Additionally, RAB27A and –B, two small GTPases involved in exosome release control were targeted. GW4869 inhibited HBV virion release, causing their accumulation along with SVPs in hepatocytes. This triggered cellular endoplasmic reticulum (ER) stress, leading to AKT-MTOR signaling pathway inactivation. GW4869 treatment increased autophagosome formation and impaired autophagic degradation by blocking autophagosome-lysosome fusion. Consequently, HBsAg is increasingly localized to autophagosomes and late endosomes/multivesicular bodies (MVBs). Silencing nSMase yielded consistent results. Similarly, RAB27A silencing inhibited HBV virion and SVP secretion, causing their accumulation within hepatoma cells. Notably, GW4869 treatment, as well as RAB27A and -B silencing, increased the presence of LC3⁺CD63⁺HBsAg⁺ complexes.

Conclusion: Our results demonstrate the involvement of the autophagosome-late endosome/MVB-exosome axis in regulating HBV production and release, highlighting amphisomes as a potential platform for HBV release.