乙脑病毒包膜蛋白中突破血脑屏障的关键氨基酸位点鉴定及机制研究

Japanese encephalitis virus (JEV) is neurotropic and mechanisms of crossing the blood-brain barrier (BBB) to establish an infection in brain have been hotspots in the field. JEV envelope protein E located on the surface of virus particle is considered to contain the ligand for virus entering the cells. However, the mechanism behind the amino acid composition of ligands and brain cell infection remains unknown. Our previous studies on comparison of the encephalitis-causing virulent strain SA14 with the attenuated strain SA14-14-2 showed that EGFR was a key molecule for JEV entry into host cells and the cytopathic effect of SA14 was significantly stronger than SA14-14-2 on the human brain microvascular endothelial cells. The replacement of differential amino acids in envelope protein E region of the virulent strain to the attenuated strain made the mutant strains lose the cytopathic effect in host cells. Associated works have been published. Based on our findings, we hypothesize that the envelope protein E region is critical for JEV breaking through the blood-brain barrier. This applying project plans to further continue the work by the construction of a series of envelope protein E mutants on the basis of SA14-14-2. And then utilize cell model, animal (mouse) model and structural biology methods to analyze the relationship between each mutant in disruption of blood-brain barrier and pathology of brain cells, which helps identify the key amino acids for JEV breaking through the blood-brain barrier and explore its mechanism. Therefore, this project helps not only determine the function of key amino acids of JEV envelope protein in crossing the blood-brain barrier, but also provide the potential for the prevention of JEV infection and the development of anti-JEV drugs.

乙脑病毒（JEV）具有脑神经细胞嗜性，其突破血脑屏障引起乙型脑炎的机制是领域研究热点。包膜蛋白E位于JEV表面，被认为含有病毒入胞配体，但配体的氨基酸组成及入侵脑细胞机制仍不清楚。我们前期研究发现：表皮生长因子受体是介导JEV细胞入侵的关键分子；JEV强毒株SA14对脑微血管内皮细胞致病效应显著强于减毒株SA14-14-2；将强毒株包膜蛋白E中差异氨基酸突变为减毒株后，强毒株即失去细胞致病能力；相关工作已发表系列论文。本课题拟在此基础上，以JEV突破血脑屏障为切入点，以减毒株为起始材料，构建一系列包膜蛋白E突变的毒力增强株，采用细胞/小鼠感染模型及结构生物学方法，分析突变株的血脑屏障结构破坏及脑细胞感染等表型变化，从而鉴定出JEV突破血脑屏障的关键氨基酸位点及其作用机制。课题的立项有助于确定JEV突破血脑屏障的结构基础，还可为JEV防治药物研发提供新靶点。

流行性乙型脑炎病毒（JEV）具有神经细胞嗜性，突破血脑屏障是其致病的先决条件。本课题组前期利用能引起脑炎的JEV强毒株SA14和不引起脑炎的JEV减毒株SA14-14-2进行比较研究，证明了影响JEV突破血脑屏障的关键位点极有可能存在于包膜蛋白E中。本课题在前期工作基础上，构建包膜蛋白E突变株，采用细胞和小鼠模型，分析JEV入侵、感染脑微血管内皮细胞的机制。.本课题构建了JEV疫苗株SA14-14-2感染性克隆。首先将JEV减毒活疫苗株SA14-14-2分两段合成，继而通过酶切连接的方法将上述两片段与载体pACNR进行连接，构建出pACNR-SA14-14-2全长cDNA质粒。通过酶切鉴定和测序鉴定，SA14-14-2全长 cDNA 质粒模板成功构建，顺利得到拯救病毒，经免疫荧光试验检测到拯救病毒E蛋白、NS1病毒蛋白和NS3病毒蛋白的表达。拯救病毒与亲本病毒的空斑大小与形态类似，同时具有类似的增殖特性。小鼠感染试验显示，拯救病毒与亲本病毒对小鼠均无神经毒力和神经侵袭力。本研究成功构建了JEV减毒活疫苗株SA14-14-2的感染性克隆，获得拯救病毒，为研究JEV的致病机制和减毒机制提供了基础。.本课题利用人膜转运相关分子siRNA文库，高通量筛选出参与JEV感染原代人脑微血管内皮细胞（HBMEC）的关键宿主分子。进一步系统分析证实JEV入侵HBMEC依赖于caveolin-1介导的内吞途径（小窝途径），而不需要网格蛋白介导的内吞途径参与。机制分析显示，JEV的感染激活了一条Src/ezrin/caveolin-1通路，并诱导形成了p-Src/p-ezrin/p-caveolin-1信号复合物。更为重要的是，我们发现ezrin不仅是JEV入侵中组织肌动蛋白重组的重要分子，也是Src介导caveolin-1活化进而促进JEV通过小窝途径入侵HBMEC的关键因素。动物水平研究还发现针对Src（PP2）和ezrin（NSC668394）的抑制剂均能降低感染小鼠大脑病毒载量，减轻脑组织病理损伤并显著提高存活率。这不仅加深了对JEV穿越血脑屏障机制的理解，也为抗JEV感染提供了新的策略。