Hepatitis B virus hijacks TSG101 to facilitate egress via multiple vesicle bodies.
Hepatitis B virus hijacks TSG101 to facilitate egress via multiple vesicle bodies.
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DOI：
10.1371/journal.ppat.1011382
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发表时间：
2023-05
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
作者：
B型肝炎病毒（HBV）慢性感染2.96亿人，目前尚无治愈方法。作为病毒生命周期的重要环节，HBV的逃逸机制至今仍不清楚。利用蛋白质组学方法鉴定衣壳蛋白（HBc）相关的宿主因子，并通过siRNA筛选，我们发现了肿瘤易感基因101（TSG 101）。在HBV产生细胞、HBV感染细胞和HBV转基因小鼠中敲低TSG 101可抑制HBV释放。免疫共沉淀和定点突变显示TSG 101中的VFND基序和HBc中的Lys-96泛素化是TSG 101-HBc相互作用所必需的。体外泛素化实验表明，UbcH 6和NEDD 4分别是潜在的E2泛素结合酶和E3连接酶，催化HBc泛素化。HBc中的PPAY基序和NEDD 4中的Cys-867是HBc泛素化、TSG 101-HBc相互作用和HBV外出所必需的。透射电子显微镜证实，TSG 101或NEDD 4敲低减少了多泡体（MVB）中的HBV颗粒计数。我们的工作表明TSG 101对NEDD 4泛素化HBc的识别对于MVB介导的HBV外出至关重要。HBV病毒体组装起始于核衣壳转运至MVB表面，然后通过内体分选复合物与HBV包膜蛋白接触时通过转运所需的内体分选复合物（ESCRT）复合物出芽成MVB。然而，目前尚不清楚宿主因子通过哪些以及如何识别HBV病毒粒子并将其分类为MVB以启动外出途径。这项研究表明，TSG 101通过其VFND序列识别泛素化的HBc，并使HBV进入MVBs的出口路线。此外，我们发现，在HBc的Lys-96残基和PPAY基序是必不可少的HBc和TSG 101的桥接。我们还证明了内体E3泛素连接酶NEDD 4催化HBc泛素化，并且HBc中的PPAY基序是NEDD 4募集所必需的。总之，我们的研究结果提供了深入了解HBV病毒粒子如何被分选成MVB流出的分子机制。
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. 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.
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影响因子：
3.7
作者：
Stieler JT;Prange R
通讯作者：
Prange R
DOI：
10.1074/mcp.m114.041012
发表时间：
2015-01
期刊：
Molecular & cellular proteomics : MCP
影响因子：
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