The RNA Helicase DHX16 Provides Specific Viral RNA for RIG-I to Trigger Innate Antiviral Immunity. The RNA Helicase DHX16 Provides Specific Viral RNA for RIG-I to Trigger Innate Antiviral Immunity

Type-I interferon (IFN-I) is essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses, however few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue and identified the DEAH-box RNA helicase DHX16 as a factor that enhances IFN-I production. Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3 Ub ligase TRIM6. DHX16 recognizes a signal in influenza segments that undergo splicing and requires its intact RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a potentiator of RIG-I-mediated IFN-I production and recognizes a mechanism for activation of antiviral immunity requiring unanchored poly-Ub and the viral RNA recognition activity of DHX16 Overall design: Next Generation Sequencing of human A549 lung cells treated with either non-targeting control or DHX16 targeting siRNA and either mock or influenza A virus infected (MOI=1) for 24 hours.

I型干扰素（Type-I interferon, IFN-I）是建立抗病毒天然免疫所必需的关键因子。未锚定（游离）多聚泛素（unanchored (or free) polyubiquitin, poly-Ub）已被证实可调控I型干扰素应答，但目前已知的未锚定多聚泛素互作蛋白仍屈指可数。为在生理环境中鉴定受未锚定多聚泛素调控的蛋白因子，我们开发了一种从肺组织中分离未锚定多聚泛素的方法，并鉴定出DEAH盒RNA解旋酶DHX16可作为增强I型干扰素产生的因子。在细胞水平与活体动物中沉默DHX16，均会削弱机体针对甲型流感病毒的I型干扰素应答。DHX16依赖的I型干扰素产生过程，需要视黄酸诱导基因蛋白I（RIG-I）以及由E3泛素连接酶TRIM6合成的未锚定K48连接型多聚泛素参与。DHX16可识别流感病毒分段基因组中发生剪接的区段内的信号序列，且需依赖其完整的RNA解旋酶基序，才能与特定病毒RNA发生直接的高亲和力结合。本研究确立了DHX16作为RIG-I介导的I型干扰素产生增强剂的地位，并揭示了一种依赖未锚定多聚泛素与DHX16病毒RNA识别活性的抗病毒免疫激活机制。总体实验设计：对分别转染非靶向对照siRNA或靶向DHX16的siRNA的人源A549肺细胞，同时设置模拟感染组与甲型流感病毒感染组（感染复数MOI=1，感染24小时）进行下一代测序（Next Generation Sequencing）。