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.

中文翻译：

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乙型肝炎病毒劫持 TSG101 以促进通过多个囊泡体排出。

乙型肝炎病毒 (HBV) 长期感染 2.96 亿人，并且无法治愈。作为病毒生命周期的一个重要步骤，HBV 排出的机制仍未得到充分阐明。通过蛋白质组学方法鉴定衣壳蛋白 (HBc) 相​​关宿主因子和 siRNA 筛选，我们发现了肿瘤易感基因 101 (TSG101)。在 HBV 产生细胞、HBV 感染细胞和 HBV 转基因小鼠中敲低 TSG101 可抑制 HBV 释放。免疫共沉淀和位点诱变表明，TSG101 中的 VFND 基序和 HBc 中的 Lys-96 泛素化对于 TSG101-HBc 相互作用至关重要。体外泛素化实验表明，UbcH6 和 NEDD4 是潜在的 E2 泛素结合酶和 E3 连接酶，分别催化 HBc 泛素化。HBc 泛素化需要 HBc 中的 PPAY 基序和 NEDD4 中的 Cys-867，TSG101-HBc 相互作用和 HBV 排出。透射电子显微镜证实，TSG101 或 NEDD4 敲低可减少多泡体 (MVB) 中的 HBV 颗粒数。我们的工作表明，TSG101 对 NEDD4 泛素化 HBc 的识别对于 MVB 介导的 HBV 排出至关重要。