Discrimination of nonself by innate immune receptors

先天免疫系统作为第一道防线，刺激宿主对微生物和病毒感染的反应。先天免疫系统的特异性受体检测病原体中的分子模式以诱导干扰素和促炎细胞因子的产生。干扰素诱导数百个基因的表达以建立抗病毒状态并调节适应性免疫，进一步加强宿主对感染的反应。RIG-I（视黄酸诱导基因- I）是一个受体家族的原型成员，其在区分细胞质中的病毒和细胞RNA中起关键作用。RIG-I受体家族识别多种重要的人类病毒，包括流感病毒、丙型肝炎病毒、登革热病毒、西尼罗河病毒、呼吸道合胞病毒、呼肠孤病毒和埃博拉病毒。我们研究的一个主要目标是了解RIG-I识别称为PAMP（病原体相关分子模式）的感染病原体的非典型特征的热力学，动力学和结构机制。 RIG-I蛋白由两个N-末端CAspase募集结构域（CARD）、一个中心DExD/H RNA解旋酶结构域和一个C-末端阻遏结构域（RD）组成。为了了解RIG-I的不同结构域之间的协同作用，以及ATP水解对RIG-I活化的贡献，我的实验室确定了与dsRNA和不可水解的ATP类似物结合的人RIG-I的RNA结合部分（解旋酶-RD）的结构，从而对RIG-I活化产生了新的见解。主要通过RIG-I在细胞质中检测具有5 '-三磷酸（ppp）的RNA。认为自身RNA如mRNA不被RIG-I识别，因为5 ′ ppp通过向5 ′端核苷酸核糖（Cap-1）添加7-甲基鸟苷（m7 G）（Cap-0）和2 ′-O-甲基而加帽。我们为加帽和2 '-O-甲基化在逃避RIG-I识别中的确切作用提供了结构和机制依据。令人惊讶的是，Cap-0和5 'ppp dsRNA以几乎相同的亲和力结合RIG-I，并以相似的程度激活RIG-I的ATP酶和细胞信号传导应答。RIG-I与带有5 'OH、5' ppp和Cap-0的dsRNA的复合物的三种晶体结构表明，RIG-I可以将m7 G帽容纳在通过解旋酶中的构象变化产生的空腔中，而不干扰ppp相互作用。相比之下，Cap-1修饰通过涉及H830残基的机制消除RIG-I信号传导，我们已经证明这对于区分Cap-0和Cap-1 RNA至关重要。 RIG-I的ATP酶活性在RNA识别和激活中起作用，但其机制尚不清楚。使用瞬态动力学，我们阐明了RIG-I活化和RNA辨别的ATP酶驱动的动力学校正机制，类似于DNA聚合酶、核糖体和T细胞受体（Devarkar等人，Mol. Cell 2018）。Singleton-Merten综合征（SMS）患者患有一种罕见的自身免疫性疾病，由RIG-I中的突变（C268 F或E373 A）引起，该突变在没有感染的情况下激活其信号传导。ATP结合促进dsRNA接合，但有趣的是，突变体使RIG-I混杂，解释了组成性信号传导。ATP水解比病毒dsRNA更快地解离自身RNA，但更重要的是，通过易位驱动RIG-I寡聚化。RIG-I从dsRNA末端定向易位到茎区，并且5 ppp末端抑制易位以提供用于穿线和构建信号传导活性寡聚复合物的机制。此外，使用氢/氘交换，提出了SMS突变体对RIG-I校正的失调的机制模型，其最终导致细胞RNA的不正确识别（Zheng等人，Nature Commun. 2018年）。

The innate immune system acts as the first line of defense to stimulate the hosts response against microbial and viral infections. Specific receptors of the innate immune system detect molecular patterns in the pathogen to induce the production of interferon and proinflammatory cytokines. Interferon induces the expression of hundreds of genes to establish an antiviral state and modulate adaptive immunity, further strengthening the hosts response against infection. RIG-I (Retinoic acid Inducible Gene - I) is the prototypical member of one family of receptors that plays a critical role in discriminating between viral and cellular RNA in the cytoplasm. The RIG-I family of receptors recognizes a variety of of important human viruses, including Influenza, Hepatitis C, Dengue, West Nile, Respiratory Syncytial, Reovirus, and Ebola. A major goal of our research is to understand the thermodynamic, kinetic, and structural mechanisms by which RIG-I recognizes atypical features of infecting pathogens called PAMPs (Pathogen Associated Molecular Patterns). The RIG-I protein consists of two N-terminal CAspase Recruitment Domains (CARDs), a central DExD/H RNA helicase domain followed by a C-terminal repression domain (RD). To understand synergy among the different domains of RIG-I for RNA binding and the contribution of ATP hydrolysis to RIG-I activation, my laboratory determined the structure of the RNA binding portion of human RIG-I (helicase-RD) bound to both dsRNA and a non-hydrolysable ATP analogue, yielding new insights into RIG-I activation. RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by RIG-I. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5'ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2'-O-methyl group to the 5'-end nucleotide ribose (Cap-1). We provided structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp dsRNAs bind to RIG-I with nearly identical affinities and activate RIG-I's ATPase and cellular signaling response to similar extents. Three crystal structures of RIG-I complexes with dsRNAs bearing 5'OH, 5'ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we have shown is crucial for discriminating between Cap-0 and Cap-1 RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient-state kinetics, we elucidated the ATPase-driven kinetic proofreading mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T cell receptors (Devarkar et al. Mol. Cell 2018). Patients with Singleton-Merten syndrome (SMS) have a rare autoimmune disease caused by a mutation (either C268F or E373A) in RIG-I that active it for signaling in the absence of infection. ATP binding facilitates dsRNA engagement but, interestingly, the mutants make RIG-I promiscuous, explaining the constitutive signaling. ATP hydrolysis dissociates self-RNAs faster than viral dsRNA but, more importantly, drives RIG-I oligomerization through translocation. RIG-I translocates directionally from the dsRNA end into the stem region, and the 5ppp end throttles translocation to provide a mechanism for threading and building a signaling-active oligomeric complex. Furthermore, using hydrogen/deuterium exchange, mechanistic models were presented for dysregulation of RIG-I proofreading by SMS mutants that ultimately result in the improper recognition of cellular RNAs (Zheng et al. Nature Commun. 2018) .