呼吸道合胞病毒（RSV）感染与儿童急性脑病和中枢神经系统症状的关系越来越受重视。我们前期研究发现，TLR4和核仁素能结合RSV的F蛋白，协同感染中枢神经元。但两受体与F蛋白的作用机制尚不清楚。TLR4的TIR结构域和核仁素GAR结构域被认为是蛋白质相互作用结构域，对介导病毒入胞及感染具重要作用。我们的预实验结果显示，TIR和GAR结构域存在共定位并可能与F蛋白相互作用。因此我们设想，TIR和GAR结构域及与F蛋白间互作可能协同介导了RSV感染，但其具体机制还待进一步研究。本项目拟构建TIR和GAR结构域的表达和缺失慢病毒，运用CO-IP、pull-down、激光共聚焦、流式细胞术等方法和手段，在细胞和分子水平上探讨TIR和GAR结构域对RSV感染致细胞损伤的协同作用，以阐明其在RSV感染神经元中的互作机理。对制备晶体复合物寻找相互作用位点，进一步完善F蛋白与受体相互作用理论，具有重大意义。

The relationship between respiratory syncytial virus (RSV) infection and childhood acute encephalopathy as well as central nervous system symptoms has been paid more and more attentions. Previous research by our group showed that TLR4 and nucleolin can recognize the F protein of RSV and synergistically participate in the viral infection of central neurons. But the mechanism of interaction that the two receptors of TLR4 and nucleolin binds to the F protein remains unclear. The TIR domain of TLR4 and the GAR domain of nucleolin are considered to be the interaction domains of protein and play an important role in mediating viral entry into cells and infecting host cells. Our preliminary confocal and immunoprecipitation experiments detected by coomassie brilliant blue staining also found that after RSV infection of neurons the TIR domain expressed by lentiviral transfection labeled with tag protein antibody can pull down the nucleolin and F proteins, and the GAR domain expressed by lentiviral transfection labeled with another tag protein antibody also pulls down the TLR4 and F proteins. There exists a co-localization between TIR domain and GAR domain and a possible interaction between TIR domain and GAR domain with F protein. So we hypothesized that the interaction of the TIR and GAR domains and their combination with F protein may synergistically mediate the RSV infection. However, the mechanisms how they mediate RSV infection and cellular damage remains to be further studied. In the current project we intend to construct the lentiviruses of expression and deletion of TIR and GAR domain, respectively. The immunoprecipitation, GST pull-down, laser confocal, flow cytometry, Western-blot, ELISA as well as other methods were used to investigate the synergistic effect of TIR4 TIR domain and nucleolin GAR domain on RSV infection and its influence on cell damage at the cellular and molecular levels, in order to elucidate its interaction mechanism in RSV-infected central neurons. It is of great significance to prepare complex crystal structure and find its interaction sites, as well as further improve the interaction theory between RSV F protein and its receptors.