乙型脑炎病毒NS2B蛋白激活NLRP3炎性小体的作用研究

Japanese encephalitis virus (JEV) infection causes severe encephalitis by invading the central nervous system and inducing excessive neuroinflammation by activating the microglia which subsequently results in the production of inflammatory cytokines. Recent evidence has unveiled that JEV infection of microglia triggers the NLRP3 inflammasome complex ensuing the release of IL-1β and IL-18 which play important roles in inflammatory diseases. However, the mechanism by which component of JEV activates the NLRP3 inflammasome is unknown. Viroporins are small, hydrophobic virus-encoded proteins that increase membrane permeability to ions or small molecules by the formation of the ion channel pores. Viroporin-induced disruptions of ionic homeostasis in the cytoplasm play important roles in the activation of NLRP3 inflammasome. By analyzing the sequences using secondary structure algorithms, we find that the sequence and structure of nonstructural protein NS2B conform to the general features of viroporin. Our previous study demonstrated that NS2B is involved in viral particle assembly process in which other viroporins involve. These results strongly suggest that NS2B is the potential viroporin of JEV, and therefore plays important roles in NLRP3 stimulation during virus infection. In this project, we will use the mouse microglia as the working model, to identify whether the nonstructural protein NS2B which has the potential viroporin activity, is essential in triggering the NLRP3 inflammasome during JEV infection. Further, the effects of NS2B on the cellular ion balance of microglia, the ion channel property of NS2B, and the effects of channel inhibitors on IL-1β release will be sequentially examined to investigate the mechanism of NS2B induced NLRP3 inflammasome activation. This project aims to discover which component of JEV stimulates the NLRP3 inflammasome of microglia to provide theoretical basis for development of antiviral drugs that target viral protein and inhibit the neuroinflammation.

乙型脑炎病毒（JEV）感染中枢神经系统，刺激小胶质细胞释放大量炎症因子，引起过度的炎症反应，是其引起病毒性脑炎的主要原因。JEV激活小胶质细胞NLRP3炎性小体引起强炎症因子IL-1β和IL-18的释放，但引起NLRP3激活的病毒组分仍不清楚。蛋白质序列分析和我们的前期研究结果表明JEV非结构蛋白NS2B具有潜在孔形成蛋白（viroporin）的活性，而viroporin通过在细胞膜上形成离子通道造成胞内离子紊乱是其他RNA病毒激活NLRP3的重要机制，因此本项目以小胶质细胞为模型，研究NS2B在JEV激活NLRP3炎性小体中的作用。在此基础上，通过检测NS2B的离子通道活性以及离子通道抑制剂对小胶质细胞IL-1β释放的影响来阐明NS2B激活NLRP3炎性小体的机制。本项目旨在揭示JEV刺激宿主NLRP3炎性小体的病毒成分，为将来研发靶向病毒蛋白的抑制神经炎症反应的抗病毒药物提供理论依据。

乙型脑炎病毒（JEV）感染中枢神经系统，刺激小胶质细胞释放大量炎症因子，引起过度的炎症反应，是其引起病毒性脑炎的主要原因。已有研究报道JEV能够激活小胶质细胞NLRP3炎性小体引起强炎症因子的释放，但引起NLRP3激活的病毒组分仍不清楚。病毒孔蛋白（viroporin）通过在细胞膜上打孔造成胞内离子紊乱是其他RNA病毒激活NLRP3的重要机制。蛋白质序列分析和我们的前期研究结果表明JEV非结构蛋白NS2B具有潜在viroporin的活性。. 本项目的预期目标是阐释NS2B在JEV激活小胶质细胞NLRP3炎性小体中的作用及其机制，以揭示JEV刺激宿主NLRP3炎性小体的病毒成分，为将来研发抗病毒药物提供依据。在项目执行过程中，我们将JEV病毒感染小鼠小胶质细胞BV-2，发现病毒并不能在该细胞中有效扩增，未能成功建立JEV感染小胶质细胞的模型，因此对重点研究内容进行了调整，主要对符合viroporin活性的JEV NS2B参与病毒复制与包装的机制进行了探索。. 利用病毒学和细胞生物学手段，我们首次发现黄病毒属病毒NS2B与NS2A相互作用参与病毒包装过程，NS2B与NS4A-2K相互作用在病毒复制过程中发挥功能；通过构建NS2A带有Flag标签的JEV病毒，首次在病毒学水平验证了NS2B与NS2A的相互作用，并发现NS2B在招募病毒复制复合体形成中发挥重要功能；发现NS2B突变可能影响NS2A蛋白的稳定性，进而影响病毒的包装。以上研究成果有助于理解NS2B在病毒复制和感染中发挥的功能及其机制，为抗病毒药物筛选提供新的潜在作用靶点。