ABSTRACT

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 of ezrin, 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 of in vitro kinase 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.

摘要

日本脑炎病毒 (JEV) 仍然是全世界病毒性脑炎的主要原因。它通过尚未完全了解的致病机制穿过血脑屏障到达中枢神经系统。在这里，使用高通量 siRNA 筛选试验结合验证实验，我们发现 JEV通过小窝介导的内吞途径进入原代人脑微血管内皮细胞 (HBMEC)。的作用ezrin 是基于我们筛选的 JEV 进入的重要宿主因子，在小窝介导的 JEV 内化中进行了研究。我们观察到 HBMEC 中的 JEV 内化在很大程度上取决于 ezrin 介导的肌动蛋白细胞骨架聚合。此外，发现在 JEV 条目中需要 Src，一种通过 STRING 数据库搜索预测的蛋白质。通过各种药理抑制和免疫沉淀试验，我们发现 Src、ezrin 和 caveolin-1 在 JEV 感染期间依次被激活并形成复合物。的组合在体外激酶测定和亚细胞分析表明，埃兹蛋白为SRC-小窝蛋白-1的相互作用是必不可少的。体内，Src 和 ezrin 抑制剂都保护 ICR 乳鼠免受 JEV 诱导的死亡和减少的小鼠脑病毒载量。因此，JEV 进入 HBMEC 需要激活 Src-ezrin-caveolin-1 信号轴，这为限制 JEV 感染提供了潜在的目标。