Ezrin is essential for the entry of Japanese encephalitis virus into the human brain microvascular endothelial cells
Ezrin is essential for the entry of Japanese encephalitis virus into the human brain microvascular endothelial cells
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Ezrin对于日本脑炎病毒进入人脑微血管内皮细胞至关重要
DOI：
10.1080/22221751.2020.1757388
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发表时间：
2020-01-01
影响因子：
13.2
通讯作者：
Qi, Zhongtian
中科院分区：
文献类型：
--
作者：
Liu, Yan-Gang;Chen, Yang;Qi, Zhongtian
日本脑炎病毒（JEV）仍然是世界范围内病毒性脑炎的主要病因。它在穿过血脑屏障后到达中枢神经系统，其致病机制尚不完全清楚。本研究通过高通量siRNA筛选实验结合验证实验，发现乙脑病毒通过小泡介导的内噬途径进入人脑微血管内皮细胞（HBMEC）。根据我们的筛选，fezrin是乙脑病毒进入的重要宿主因子，研究了fezrin在小泡介导的乙脑病毒内化中的作用。我们观察到乙脑病毒在HBMEC中的内化在很大程度上依赖于ezrin介导的肌动蛋白细胞骨架聚合。此外，通过STRING数据库搜索预测的蛋白Src在乙脑病毒的进入中是必需的。通过多种药理抑制和免疫沉淀实验，我们发现Src、ezrin和caveolin-1在乙脑病毒感染期间依次被激活并形成复合物。结合vitrokinase assay和亚细胞分析表明ezrin是Src-caveolin-1相互作用的关键。在体内，Src和ezrin抑制剂都能保护ICR哺乳小鼠免受jev诱导的死亡，并降低小鼠脑病毒载量。因此，JEV进入HBMEC需要激活Src-ezrin-caveolin-1信号轴，这为限制JEV感染提供了潜在的靶点。
Japanese encephalitis virus (JEV) remains the predominant cause of viral encephalitis worldwide. It reaches the central nervous system upon crossing the blood-brain barrier through pathogenic mechanisms that are not completely understood. Here, using a high-throughput siRNA screening assay combined with verification experiments, we found that JEV enters the primary human brain microvascular endothelial cells (HBMEC) through a caveolae-mediated endocytic pathway. The role ofezrin, an essential host factor for JEV entry based on our screening, in caveolae-mediated JEV internalization was investigated. We observed that JEV internalization in HBMEC is largely dependent on ezrin-mediated actin cytoskeleton polymerization. Moreover, Src, a protein predicted by a STRING database search, was found to be required in JEV entry. By a variety of pharmacological inhibition and immunoprecipitation assays, we found that Src, ezrin, and caveolin-1 were sequentially activated and formed a complex during JEV infection. A combination ofin vitrokinase assay and subcellular analysis demonstrated that ezrin is essential for Src-caveolin-1 interactions.In vivo, both Src and ezrin inhibitors protected ICR suckling mice against JEV-induced mortality and diminished mouse brain viral load. Therefore, JEV entry into HBMEC requires the activation of the Src-ezrin-caveolin-1 signalling axis, which provides potential targets for restricting JEV infection.