受体酪氨酸激酶应答促进乙型脑炎病毒入侵的分子机制

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus and one of the most important flaviviruses in medical and veterinary fields. The fatality rate and disability rate due to Japanese encephalitis are high. Our knowledge on how the host cells signal pathway response to take effects on JEV infection is very limited. Receptor tyrosine kinases (RTKs) are the high-affinity cell surface kinases family, which are the key molecules that deliver the signal into the cell. The complex signaling network triggered by RTKs leads to regulation of cell growth, migration, metabolism, and differentiation. We have screened the RTKs siRNA library by using the replicon virus particles and found that platelet-derived growth factor receptorβ (PDGFRβ) was involved in the JEV entry. The JEV infection was inhibited by knockdown of PDGFRβ or by using PDGFRβ inhibitor. Based on these, we will study the activation and distribution of PDGFRβ when infected with JEV. Moreover, the mechanism underlying the complex signaling network triggered by RTKs on regulating the kinases phosphorylation, endocytosis, actin polymerization and endosomal trafficking will be studied. Then the antiviral effect of Imatinib, the inhibitor of PDGFRβ, will be examined. An adaptive mutation will be established by serial passaging of JEV in PDGFRβknockout cells, and the target sites will be indentified by sequencing. Our study will provide a new insight into the role of RTKs on virus entry and contribute a useful experimental data to develop therapeutic method to treat JEV-related diseases.

乙型脑炎病毒（JEV）是感染人类中枢神经系统最常见的虫媒病原之一，引起的乙型脑炎致死致残率较高。目前对JEV的入侵机制，尤其是在与细胞相互识别、接触的早期阶段，细胞如何通过信号转导调控、应对JEV入侵的机制不明。受体酪氨酸激酶（RTKs）是一类锚定在细胞膜上的激酶家族，是将胞外信号传递给胞内的关键分子，与多种信号通路相关。我们运用复制子病毒颗粒对RTKs siRNA文库筛选，发现血小板衍生生长因子受体β（PDGFRβ）与JEV入侵相关，抑制其表达和活性均能有效抑制JEV感染。在此基础上，本项目拟考察JEV入侵对PDGFRβ活化和分布的影响；重点考察以PDGFRβ为核心的信号网络如何通过调控激酶磷酸化、内吞、微丝聚集及囊泡运输来应答JEV入侵；通过筛选适应性突变株，明确PDGFRβ的作用位点；考察PDGFRβ抑制药物的抗JEV功效。为抗JEV研究提供潜在靶标，为探索其它病毒入侵机制提供借鉴。

乙型脑炎病毒（JEV）是感染人类中枢神经系统最常见的虫媒病原之一，引起的乙型脑炎致死致残率较高。目前对JEV的入侵机制，尤其是在与细胞相互识别、接触的早期阶段，细胞如何通过信号转导调控、应对JEV入侵的机制不明。受体酪氨酸激酶（RTKs）是一类锚定在细胞膜上的激酶家族，是将胞外信号传递给胞内的关键分子，与多种信号通路相关。我们运用复制子病毒颗粒对RTKs siRNA文库筛选，发现血小板衍生生长因子受体β（PDGFRβ）与JEV入侵相关，抑制其表达和活性均能有效抑制JEV感染。考察JEV感染对PDGFRβ磷酸化的影响，发现JEV感染引发PDGFRβ磷酸化/二聚，PDGFRβ胞内域Y771和Y1009发生磷酸化，触发胞内信号通路级联活化。建立PDGFRβ敲除细胞株，发现敲除PDGFRβ抑制JEV感染，转染PDGFRβ至敲除细胞可回补JEV感染性。进一步考察PDGFRβ抑制剂伊马替尼的抗病毒活性。发现伊马替尼在细胞水平和动物水平均能有效抑制JEV感染。体外实验发现伊马替尼能抑制JEV对不同细胞的感染，IC50为4.987至26.17 µM。在动物试验中发现伊马替尼能显著降低JEV引起的小鼠死亡率；伊马替尼通过降低小鼠脑组织中病毒滴度、减轻病理损伤而降低JEV感染致死率。以上研究成果为阐述JEV感染机制提供借鉴，为研发抗JEV药物提供潜在靶标，为应对JEV及其它黄病毒的爆发流行提供应急药物储备。