流感病毒内吞进入宿主细胞主要是通过网格蛋白介导的方式，而涉及高致病性H5N1病毒内吞相关宿主因子仍未见报道。G蛋白偶联受体（GPCR）是一大类蛋白受体统称，已报道参与艾滋病、狂犬病、埃博拉等病毒内吞过程。GPCR在高致病性H5N1病毒的内吞过程中发挥着怎样的作用尚未见报道。因此，本研究将人工合成的针对380个GPCR基因的siRNA转染到A549细胞，再感染H5N1 NA-Venus病毒，通过高内涵成像系统扫描图像，并用Columbus进行数据分析。最终筛选到一些参与流感病毒内吞过程中的GPCR分子，阐明其在病毒复制周期中作用的关键环节；同时，探究GPCR分子如何调控下游信号分子（AP2复合体、GRKs和β-arrestins）来启动流感病毒内吞。本项目的实施将深化对GPCR参与流感病毒复制周期调控分子机制的科学认知，同时也为流感病毒的药物治疗提供新思路和新靶点。

The majority of the influenza virus particles enter into host cells through clathrin-mediated endocytosis. Although many host factors have been identified to participate in the process of low pathogenic influenza virus endocytosis, host factors associated with highly pathogenic H5N1 virus endocytosis have not been reported. GPCR constitutes a large protein family of membrane receptors, which has been reported to be involved in the endocytosis of HIV, RABV, Ebola virus and Marburg virus. So how does GPCR play role in the endocytosis of the highly pathogenic H5N1 virus? In previous work, we synthesized the 1140 siRNA of 380 GPCR genes (three independent siRNA for each gene), which was individually transfected into A549 cells in 384 well for 48 h, and then infected with the H5N1 NA-Venus virus. Images were captured with an Operetta high-content image system, analyzed by Columbus software. Finally, we will obtain some positive GPCR. On this basis, we will carry out functional experiments to determine the candidate GPCR effect on the specific stages of viral infection; Meanwhile, we will further illuminate how the positive GPCR molecules regulate downstream signaling molecules (AP2 complex, GRKs and β-arrestins) to initiate the endocytosis of influenza virus. The implementation of this project will deepen the understanding on the molecular mechanisms of GPCR functions in the regulation of influenza virus life cycle, and will provide potentially new anti-viral targets for influenza virus.