冠状病毒是一类引起人类呼吸道感染的重要病原体，其中的SARS病毒和MERS病毒的感染能导致严重肺炎，危及生命。许多冠状病毒通过内吞途径侵染宿主细胞，而内吞在细胞中受到多个因子调控，其中包括磷脂肌醇。但除了PI(4,5)P2，大多数磷脂肌醇在冠状病毒S蛋白介导的入侵中的作用并不清楚，尤其是PI(3,5)P2和PI(3,4)P2。在我们的前期研究中，通过特异性的抑制剂和siRNA敲低实验，我们初步发现PI(3,5)P2的合成酶PIKfyve以及效应分子TRPML1和TPC2在多个冠状病毒S蛋白介导入侵中起着重要作用。本项目将在此基础上，利用不同磷脂肌醇特异的抗体和biosensor，通过超高分辨率显微镜、全内反射荧光显微镜以及活细胞成像系统等细胞生物学技术手段，进一步研究PI(3,4)P2和PI(3,5)P2在冠状病毒病毒入侵中的作用和机制，为新型抗冠状病毒药物的研发提供理论基础。

Coronaviruses (CoVs) are important pathogens causing varieties of diseases among human and animals, of which Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) can cause severe pneumonia in human leading to death. Most of CoVs enter the host cells through endocytosis, which is highly regulated by various phosphatidylinositols. However, except for PI(4,5)P2, the functional roles of other phosphatidylinositols in coronavirus entry remains unknown, especially PI(3,4)P2 and PI(3,5)P2. Using siRNAs and inhibitors, we recently found that PIKfyve, the major PI(3,5)P2 synthetase, and TRPML1 and TPC2, two important PI(3,5)P2 effectors and calcium channels in lysosome, are critical for CoV S protein mediated entry through endocytosis. In this study, we propose to utilize super-resolution microscope, total internal reflection fluorescence (TIRF) microscope, and spindisck microscope with live imaging system, in combination with individual phosphatidylinositol specific antibodies and biosensors, to investigate the mechanism of how PI(3,4)P2 and PI(3,5)P2 regulate CoV entry through endocytosis. These discoveries will provide potential new targets for anti-CoV drug development.