Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
iEGG and Animal Biotechnology Center, National Chung Hsing University, Taichung, Taiwan.
Microbiol Spectr. 2023 Jun 15;11(3):e0000923. doi: 10.1128/spectrum.00009-23. Epub 2023 Apr 25.
The specifics of cell receptor-modulated avian reovirus (ARV) entry remain unknown. By using a viral overlay protein-binding assay (VOPBA) and an in-gel digestion coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we determined that cell-surface annexin A2 (AnxA2) and adhesion G protein-coupled receptor Latrophilin-2 (ADGRL2) modulate ARV entry. Direct interaction between the ARV σC protein and AnxA2 and ADGRL2 in Vero and DF-1 cells was demonstrated by proximity ligation assays. By using short hairpin RNAs (shRNAs) to silence the endogenous AnxA2 and ADGRL2 genes, ARV entry could be efficiently blocked. A significant decrease in virus yields and the intracellular specific signal for σC protein was observed in Vero cells preincubated with the specific AnxA2 and ADGRL2 monoclonal antibodies, indicating that AnxA2 and ADGRL2 are involved in modulating ARV entry. Furthermore, we found that cells pretreated with the AnxA2/S100A10 heterotetramer (A2t) inhibitor A2ti-1 suppressed ARV-mediated activation of Src and p38 mitogen-activated protein kinase (MAPK), demonstrating that Src and p38 MAPK serve as downstream molecules of cell-surface AnxA2 signaling. Our results reveal that suppression of cell-surface AnxA2 with the A2ti-1 inhibitor increased Csk-Cbp interaction, suggesting that ARV entry suppresses Cbp-mediated relocation of Csk to the membrane, thereby activating Src. Furthermore, reciprocal coimmunoprecipitation assays revealed that σC can interact with signaling molecules, lipid raft, and vimentin. The current study provides novel insights into cell-surface AnxA2- and ADGRL2-modulated cell entry of ARV which triggers Src and p38 MAPK signaling to enhance caveolin-1-, dynamin 2-, and lipid raft-dependent endocytosis. By analyzing results from VOPBA and LC-MS/MS, we have determined that cell-surface AnxA2 and ADGRL2 modulate ARV entry. After ARV binding to receptors, Src and p38 MAPK signaling were triggered and, in turn, increased the phosphorylation of caveolin-1 (Tyr14) and upregulated dynamin 2 expression to facilitate caveolin-1-mediated and dynamin 2-dependent endocytosis. In this work, we demonstrated that ARV triggers Src activation by impeding Cbp-mediated relocation of Csk to the membrane in the early stages of the life cycle. This work provides better insight into cell-surface AnxA2 and ADGRL2, which upregulate Src and p38MAPK signaling pathways to enhance ARV entry and productive infection.
细胞受体调节的禽呼肠孤病毒（ARV）进入的具体细节尚不清楚。通过使用病毒覆盖蛋白结合测定（VOPBA）和胶内消化与液相色谱-串联质谱（LC-MS/MS）联用，我们确定细胞表面膜联蛋白 A2（AnxA2）和黏附 G 蛋白偶联受体 Latrophilin-2（ADGRL2）调节 ARV 进入。通过接近连接测定，证明了 ARV σC 蛋白与 Vero 和 DF-1 细胞中的 AnxA2 和 ADGRL2 之间的直接相互作用。通过使用短发夹 RNA（shRNA）沉默内源性 AnxA2 和 ADGRL2 基因，可有效阻断 ARV 进入。在预先用特异性 AnxA2 和 ADGRL2 单克隆抗体孵育的 Vero 细胞中，观察到病毒产量和 σC 蛋白的细胞内特异性信号显著降低，表明 AnxA2 和 ADGRL2 参与调节 ARV 进入。此外，我们发现用膜联蛋白 A2/S100A10 异四聚体（A2t）抑制剂 A2ti-1 预处理的细胞抑制了 ARV 介导的Src 和 p38 丝裂原活化蛋白激酶（MAPK）的激活，表明 Src 和 p38 MAPK 作为细胞表面 AnxA2 信号的下游分子。我们的结果表明，用 A2ti-1 抑制剂抑制细胞表面 AnxA2 增加了 Csk-Cbp 相互作用，表明 ARV 进入抑制了 Cbp 介导的 Csk 向膜的重定位，从而激活了 Src。此外，相互免疫沉淀测定表明 σC 可以与信号分子、脂筏和波形蛋白相互作用。本研究为细胞表面 AnxA2 和 ADGRL2 调节 ARV 进入提供了新的见解，这种进入触发了 Src 和 p38 MAPK 信号通路，从而增强了质膜窖蛋白 1（caveolin-1）、dynamin 2 和脂筏依赖性内吞作用。通过分析 VOPBA 和 LC-MS/MS 的结果，我们确定了细胞表面 AnxA2 和 ADGRL2 调节 ARV 进入。在 ARV 与受体结合后，触发了 Src 和 p38 MAPK 信号通路，从而增加了 caveolin-1（Tyr14）的磷酸化和 dynamin 2 的表达，促进了 caveolin-1 介导和 dynamin 2 依赖性内吞作用。在这项工作中，我们证明了 ARV 通过在生命周期的早期阶段阻止 Cbp 介导的 Csk 向膜的重定位来触发 Src 的激活。这项工作更好地了解了细胞表面的 AnxA2 和 ADGRL2，它们上调了 Src 和 p38MAPK 信号通路，以增强 ARV 的进入和有效的感染。
