G蛋白偶联受体调控流感病毒入侵机制研究

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.

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。

A型流感病毒(IAV)利用许多宿主因子来完成其复制周期。本项目利用siRNA文库对G蛋白偶联受体（GPCR）家族蛋白进行筛选，最终从380个GPCR基因中筛选出9个基因（ADORA3、CXCR4、CXCR5、MC1R、FFAR2、GABRA1、NPY1R、PTAFR、SSTR1）能够明显地抑制H5N1 NA-Venus报告病毒复制。随后，我们选择游离脂肪酸受体2 (FFAR2)进行深入机制解析。我们发现，在A549或RAW 264.7细胞中下调表达FFAR2显著降低IAV复制。用FFAR2 siRNA或FFAR2通路激动剂2-(4-氯苯基)-3-甲基-n-(噻唑-2-基)丁酰胺(4-CMTB)和(S)-2-(4-氯苯基)-3,3-二甲基-n-(5-苯基噻唑-2-基)丁酰胺(Cmp58)处理A549细胞，在感染早期就能显著抑制NP蛋白入核，表明FFAR2在IAV复制周期的早期发挥作用。FFAR2下调表达对细胞膜表面唾液酸(SA)受体表达、IAV与SA受体结合以及病毒核糖核蛋白(vRNP)复合物的活性均无影响。相反，在FFAR2下调表达、4-CMTB-或Cmp58处理的A549细胞中，内化的IAV数量显著减少。进一步研究表明，FFAR2与β-arrestin1存在相互作用，β-arrestin1与AP-2复合物(AP2B1)也存在相互作用，其中AP2B1是网格蛋白介导的内吞通路接头分子。值得注意的是，siRNA敲低β-arrestin1或AP2B1均显著降低IAV复制，而敲低AP2B1或Barbadin(一种靶向β-arrestin1/AP2B1复合物的抑制剂)处理可显著降低内化的IAV数量。此外，我们发现FFAR2与三种G蛋白偶联受体(GPCR)激酶(即GRK2、GRK5和GRK6)相互作用，且这些激酶下调能够抑制IAV复制。因此，我们的研究结果表明，FFAR2信号级联对IAV内吞进入宿主细胞十分重要。因此，本项目顺利开展能够完善流感病毒与GPCR蛋白家族的调控网络，为研发新型抗病毒药物提供良好的靶标。