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Published: May 24, 2023
Yingcheng Zheng, Mengfei Wang, Sitong Li,Yanan Bu, Zaichao Xu, Guoguo Zhu, Chuanjian Wu, Kaitao Zhao, Aixin Li, Quan Chen, Jingjing Wang, Rong Hua, Yan Teng, [ ... ], Yuchen Xia
Abstract
Hepatitis B virus (HBV) chronically infects 296 million individuals and there is no cure. As an important step of viral life cycle, the mechanisms of HBV egress remain poorly elucidated. With proteomic approach to identify capsid protein (HBc) associated host factors and siRNA screen, we uncovered tumor susceptibility gene 101 (TSG101). Knockdown of TSG101 in HBV-producing cells, HBV-infected cells and HBV transgenic mice suppressed HBV release. Co-immunoprecipitation and site mutagenesis revealed that VFND motif in TSG101 and Lys-96 ubiquitination in HBc were essential for TSG101-HBc interaction. In vitro ubiquitination experiment demonstrated that UbcH6 and NEDD4 were potential E2 ubiquitin-conjugating enzyme and E3 ligase that catalyzed HBc ubiquitination, respectively. PPAY motif in HBc and Cys-867 in NEDD4 were required for HBc ubiquitination, TSG101-HBc interaction and HBV egress. Transmission electron microscopy confirmed that TSG101 or NEDD4 knockdown reduces HBV particles count in multivesicular bodies (MVBs). Our work indicates that TSG101 recognition for NEDD4 ubiquitylated HBc is critical for MVBs mediated HBV egress.
Author summary
HBV virion assembly is initiated with nucleocapsid transportation to the surface of the MVBs, then buds into MVB through the endosomal sorting complex required for transport (ESCRT) complexes on contact with the HBV envelope proteins via endosomal sorting complex. However, it is still unclear by which and how host factor(s) recognizes HBV virions and sorts them into MVBs to initiate the egress pathway. This study shows that TSG101 recognizes ubiquitinated HBc via its VFND sequence and gives access for HBV to the MVBs egress route. Furthermore, we find that the Lys-96 residue and PPAY motif in HBc are essential for bridging HBc and TSG101. We also demonstrated that the endosomal E3 ubiquitin ligase NEDD4 catalyze HBc ubiquitination and the PPAY motif in HBc is required for NEDD4 recruitment. In sum, our findings provide insight into the molecular mechanism of how HBV virions were sorted into MVBs to egress.
Yingcheng Zheng, Mengfei Wang, Sitong Li,Yanan Bu, Zaichao Xu, Guoguo Zhu, Chuanjian Wu, Kaitao Zhao, Aixin Li, Quan Chen, Jingjing Wang, Rong Hua, Yan Teng, [ ... ], Yuchen Xia
Abstract
Hepatitis B virus (HBV) chronically infects 296 million individuals and there is no cure. As an important step of viral life cycle, the mechanisms of HBV egress remain poorly elucidated. With proteomic approach to identify capsid protein (HBc) associated host factors and siRNA screen, we uncovered tumor susceptibility gene 101 (TSG101). Knockdown of TSG101 in HBV-producing cells, HBV-infected cells and HBV transgenic mice suppressed HBV release. Co-immunoprecipitation and site mutagenesis revealed that VFND motif in TSG101 and Lys-96 ubiquitination in HBc were essential for TSG101-HBc interaction. In vitro ubiquitination experiment demonstrated that UbcH6 and NEDD4 were potential E2 ubiquitin-conjugating enzyme and E3 ligase that catalyzed HBc ubiquitination, respectively. PPAY motif in HBc and Cys-867 in NEDD4 were required for HBc ubiquitination, TSG101-HBc interaction and HBV egress. Transmission electron microscopy confirmed that TSG101 or NEDD4 knockdown reduces HBV particles count in multivesicular bodies (MVBs). Our work indicates that TSG101 recognition for NEDD4 ubiquitylated HBc is critical for MVBs mediated HBV egress.
Author summary
HBV virion assembly is initiated with nucleocapsid transportation to the surface of the MVBs, then buds into MVB through the endosomal sorting complex required for transport (ESCRT) complexes on contact with the HBV envelope proteins via endosomal sorting complex. However, it is still unclear by which and how host factor(s) recognizes HBV virions and sorts them into MVBs to initiate the egress pathway. This study shows that TSG101 recognizes ubiquitinated HBc via its VFND sequence and gives access for HBV to the MVBs egress route. Furthermore, we find that the Lys-96 residue and PPAY motif in HBc are essential for bridging HBc and TSG101. We also demonstrated that the endosomal E3 ubiquitin ligase NEDD4 catalyze HBc ubiquitination and the PPAY motif in HBc is required for NEDD4 recruitment. In sum, our findings provide insight into the molecular mechanism of how HBV virions were sorted into MVBs to egress.