The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry
The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry
复制标题
G蛋白偶联受体FFAR2促进甲型流感病毒进入过程中的内化
DOI：
10.1128/jvi.01707-19
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发表时间：
2020-01-01
影响因子：
5.4
通讯作者：
Li, Chengjun
中科院分区：
文献类型：
--
作者：
Wang, Guangwen;Jiang, Li;Li, Chengjun
为了完成其复制周期，IAV劫持宿主的内吞机制来入侵细胞。然而，IAV如何内化到宿主细胞中的潜在机制仍然知之甚少，强调有必要阐明宿主因素在IAV进入细胞中的作用。在本研究中，我们确定FFAR2是低致病性和高致病性IAV有效复制的重要宿主因子。我们发现，在感染的早期阶段，FFAR2促进了IAV内化到靶细胞。在进一步研究FFAR2相关蛋白在病毒复制中的作用后，我们发现FFAR2-β-arrestin1-AP2B1信号级联对于IAV的有效内吞作用是重要的。因此，我们的发现进一步加深了我们对IAV进入宿主细胞的生物学细节的理解，并使FFAR2成为抗病毒药物开发的潜在靶点。摘要甲型流感病毒(IAV)依靠多种宿主因子完成其复制周期。在这里，我们确定游离脂肪酸受体2(FFAR2)是IAV进入宿主细胞的辅助因子。我们发现，下调FFAR2或FFAR2的表达显著减少了IAV在A549或RAW 264.7细胞中的复制。用针对FFAR2或FFAR2途径激动剂2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide(4-CMTB)和化合物58(Cmp58)[(S)-2-(4-chlorophenyl)-3，3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide]的小干扰RNA处理A549细胞，可显著抑制病毒核蛋白(NP)在感染后早期的核积聚，表明FFAR2在病毒复制周期的早期阶段发挥作用。FFAR2下调对细胞膜上唾液酸(SA)受体的表达、IAV与SA受体的结合或病毒核糖核蛋白(VRNP)复合体的活性没有影响。相反，在FFAR2基因敲除或4-CMTB或Cmp58处理的A549细胞中，内化IAV的数量显著减少。进一步的研究表明，FFAR2与β-arrestin1结合，β-arrestin1与AP-2复合体的β2亚单位(AP2B1)相互作用，AP2B1是网状蛋白介导的内吞途径的重要接头。值得注意的是，β-arrestin1或AP2B1的siRNA敲除显著削弱了IAV的复制，而AP2B1的敲除或用针对β-arrestin1/AP2B1复合体的抑制剂Barbadin处理，显著减少了内化IAV的数量。此外，我们还发现FFAR2与三个G蛋白偶联受体(GPCRK)激酶(即GRK2、GRK5和GRK6)相互作用，它们的下调抑制了IAV的复制。总之，我们的发现表明，FFAR2信号级联对于IAV有效地内吞进入宿主细胞是重要的。为了完成其复制周期，IAV劫持宿主的内吞机制以入侵细胞。然而，IAV如何内化到宿主细胞中的潜在机制仍然知之甚少，强调有必要阐明宿主因素在IAV进入细胞中的作用。在本研究中，我们确定FFAR2是低致病性和高致病性IAV有效复制的重要宿主因子。我们发现，在感染的早期阶段，FFAR2促进了IAV内化到靶细胞。在进一步研究FFAR2相关蛋白在病毒复制中的作用后，我们发现FFAR2-β-arrestin1-AP2B1信号级联对于IAV的有效内吞作用是重要的。因此，我们的发现进一步加深了我们对IAV进入宿主细胞的生物学细节的理解，并使FFAR2成为抗病毒药物开发的潜在靶点。
To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2–β-arrestin1–AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development. ABSTRACT Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with β-arrestin1 and that β-arrestin1 interacted with the β2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either β-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the β-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells. IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2–β-arrestin1–AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.