Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5′ exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2′,3′-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2′,3′-cyclic guanosine monophosphate (2',3'-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Toll 样受体 7 (TLR7) 对于识别 RNA 病毒和启动抗病毒免疫至关重要。 TLR7 包含两个识别不同 RNA 降解产物的配体结合口袋：口袋 1 识别鸟苷，而口袋 2 协调富含嘧啶的 RNA 片段。我们发现核酸内切酶 RNase T2 与 5' 核酸外切酶 PLD3 和 PLD4 一起协同生成 TLR7 的配体。具体来说，RNase T2 产生鸟苷 2',3'-环状单磷酸酯封端的 RNA 片段。 PLD 核酸外切酶活性进一步释放末端 2',3'-环单磷酸鸟苷 (2',3'-cGMP) 以接合口袋 1，并且还需要为口袋 2 生成 RNA 片段。 细胞系中的功能丧失研究原代细胞证实了 PLD 活性的关键要求。生化和结构研究表明，PLD 酶形成同二聚体，具有两个对活性很重要的配体结合位点。先前鉴定的与疾病相关的 PLD 突变体未能形成稳定的二聚体。总之，我们的数据为 TLR7 检测 RNA 片段提供了机制基础。