Journal of Biological Chemistry

A critical step in Hepatitis B virus (HBV) maturation and egress is the ubiquitination of the capsid/core protein (HBc), which enables its recognition by the endosomal sorting complex required for transport (ESCRT) machinery and recruitment to multivesicular bodies (MVBs). This study investigates the role of UBE2O, an atypical E2 ubiquitin-conjugating enzyme with intrinsic E3 ligase activity, in nucleocapsid assembly and virion egress. Loss of UBE2O in HBV-infected primary human hepatocytes (PHH) and HepG2-NTCP cells led to a reduction in viral replication, as evidenced by decreased levels of intracellular HBV DNA, pgRNA, capsids, and extracellular HBeAg. Additionally, UBE2O depletion disrupted intracellular nucleocapsid assembly and impaired the secretion of enveloped virions, but the release of naked nucleocapsids remained unaffected. In contrast, UBE2O overexpression enhanced the secretion of mature virions, whereas the expression of its enzymatically inactive mutant inhibited this process. Additionally, UBE2O mediated the monoubiquitination of hypophosphorylated cytoplasmic HBc and capsids. Subcellular localization experiments using confocal microscopy and proximity ligation assays (PLA) demonstrated that UBE2O colocalizes with capsids and ubiquitinated cargo in CD63-positive MVB compartments, indicating its involvement in the endosomal secretory pathway. Collectively, this study identifies UBE2O and its catalytic activity as key regulators of the HBV virion secretion pathway, highlighting its potential as a therapeutic target for HBV treatment.

hepatitis B virus

HBV core protein

HBV capsid

UBE2O

monoubiquitination

virion maturation

multivesicular bodies

virion egress

covalently closed circular DNA

endosomal sorting complex required for transport

hypoxanthine phosphoribosyltransferase 1

multivesicular bodies

primary human hepatocytes

proximity ligation assay

ubiquitin-fused HBc variant

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé