分选连接蛋白3调控肠道病毒感染诱导I型干扰素表达的机制研究

Hand, foot and mouth disease (HFMD) which was caused by the enterovirus infection out-broke in several regions of China in the past five years. The mortality was around 20% and most were under their age of 5. Although the pathogenesis of HFMD remains unclear, the expression changes of interferon which was induced by the host innate immune was one of the important mechanism of HFMD. TLR3 is the main receptor in innate immune to recognize the RNA virus. In order to find out new members of TLR3 signal pathway which was involved in the induction of interferon after the enterovirus infection, we identify sorting nexin 3 (SNX3) as the candidate protein through the luciferase reporter assay. The overexpression of SNX3 significantly promoted the IFN-β activation which was mediated by TLR3 pathway. In this study, with the help of the luciferase reporter assay, real-time PCR, immunoprecipitation as well as conditional knockout animal models, the IFN-β activation under the inhibition of SNX3, the interacted protein of SNX3 in TLR3 signaling pathway and the role of SNX3 against enterovirus infection will be studied. The results will help to clarify the mechanism in which TLR3 signal pathway was regulated by SNX3 and the pathogenesis of HFMD.

肠道病毒等RNA病毒感染引起的手足口病近年在我国部分地区暴发疫情，给国家和社会造成沉重负担。虽然其致病机制仍不清楚，但病毒感染后宿主天然免疫介导的干扰素（IFN）表达异常是其发病机制的研究重点。TLR3是天然免疫系统识别RNA病毒的主要受体，为了寻找新的参与TLR3信号通路调控肠道病毒感染诱导IFN表达的蛋白，课题组通过荧光素酶报告基因筛选实验鉴定并得到了候选蛋白-分选连接蛋白3（SNX3），其过量表达显著促进TLR3信号通路介导的IFN-β启动子的激活。本研究拟进一步分析抑制内源性SNX3对TLR3信号通路诱导IFN的影响、确定SNX3在TLR3信号通路的相互作用蛋白及对该蛋白功能的影响，研究SNX3在宿主对抗肠道病毒感染过程中的作用。研究结果对于阐明SNX3调控TLR3信号通路的机制、完善TLR3信号通路结构，深入剖析细胞抗病毒天然免疫反应及手足口病的致病机理具有重要意义。

为了进一步阐述申请人前期筛选到的分选连接蛋白3(SNX3)在TLR3信号通路调控肠道病毒感染诱导IFN表达过程中的作用和机制，课题组利用荧光素酶报告基因实验和实时定量PCR实验证实过量表达 SNX3 显着促进 Poly I:C 和 RNA 病毒诱导的 IFN-β 和 ISRE 的激活，而抑制内源性SNX3会减弱Poly I:C 和 RNA 病毒诱导的 IFN-β 和 ISRE 的激活。对SNX3重要功能结构域突变体和不同异构体的分析显示SNX3依赖PX结构域诱导TLR3 信号转导通路的激活。接下来我们利用实时定量PCR实验和免疫共沉淀实验确定SNX3与TLR3 信号通路下游的干扰素调节因子7（IRF7）有直接的相互作用，即SNX3通过与IRF7的相互作用调控TLR3信号通路的活性。IRF7磷酸化是其活化的标志，我们借助蛋白印迹实验证实过表达SNX3促进IRF7的磷酸化，SNX3 PX结构域保守区的突变会削弱这种促进作用。当内源性SNX3受到抑制时，IRF7的磷酸化水平也受到抑制。本项目研究结果阐明了SNX3调控TLR3信号通路的分子机制，对深入剖析细胞抗病毒天然免疫反应及手足口病的致病机理具有重要的科学意义。