Hepatitis B virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis
Hepatitis B virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis
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DOI：
10.1111/cmi.13205
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发表时间：
2020-04-01
影响因子：
3.4
通讯作者：
Blanchard, Emmanuelle
中科院分区：
文献类型：
--
作者：
Herrscher, Charline;Pastor, Florentin;Blanchard, Emmanuelle
乙型肝炎病毒 (HBV) 是全世界肝硬化和肝细胞癌的主要原因，有 2.5 亿人慢性感染。乙型肝炎病毒感染周期的许多阶段已被阐明，但乙型肝炎病毒进入的机制仍知之甚少。牛磺胆酸钠共转运多肽（NTCP）作为 HBV 受体的鉴定以及易受 HBV 感染的 NTCP 过表达肝癌细胞系的建立为研究这些机制开辟了新的可能性。我们使用 HepG2-NTCP 细胞以及各种化学抑制剂和 RNA 干扰 (RNAi) 方法来研究参与 HBV 进入的宿主细胞因素。我们发现这些细胞对 HBV 的摄取依赖于肌动蛋白细胞骨架，并且不涉及巨胞饮作用或小凹介导的内吞作用。相反，进入是通过网格蛋白介导的内吞作用途径发生的。 Pitstop-2 处理和网格蛋白重链、接头蛋白 AP-2 和 dynamin-2 的 RNA 介导的沉默 (siRNA) 可抑制 HBV 内化。我们能够通过电子显微镜 (EM) 和带有免疫金标记的冷冻电镜观察到 HBV 进入网格蛋白包被的小凹和囊泡。这些数据表明 HBV 使用网格蛋白介导的内吞途径进入 HepG2-NTCP 细胞，增加了我们对完整 HBV 生命周期的了解。
Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP-overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2-NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae-mediated endocytosis. Instead, entry occurred via the clathrin-mediated endocytosis pathway. HBV internalisation was inhibited by pitstop-2 treatment and RNA-mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP-2 and dynamin-2. We were able to visualise HBV entry in clathrin-coated pits and vesicles by electron microscopy (EM) and cryo-EM with immunogold labelling. These data demonstrating that HBV uses a clathrin-mediated endocytosis pathway to enter HepG2-NTCP cells increase our understanding of the complete HBV life cycle.