Structural and mechanistic studies of self and non-self recognition by RIG-I

描述(申请人提供)：先天免疫系统是抵御微生物和病毒感染的第一道防线。未能激发早期的先天免疫反应会导致全身性感染。大量的人类病毒，包括流感、丙型肝炎、登革热、西尼罗河病毒、呼吸道合胞病毒、呼肠孤病毒和埃博拉病毒，都是由天然免疫受体RIG-I识别的。这项建议的总体目标是了解细胞质受体RIG-I(维甲酸诱导基因-I)如何区分正常的细胞RNA和病毒RNA，以刺激宿主反应。我们合作研究的一个主要目标是了解RIG-I识别RNA中非典型病原体相关分子模式(PAMP)的热力学、动力学和结构机制。我们的RIG-I与钝端dsRNA和ATP类似物结合的晶体结构为RIG-I的激活建立了一种新的范式。这项提议的目标是使用精心设计的生化、动力学和结构研究来严格测试RIG-I激活模型。我们的初步结果表明，RIG-I受到多种方式的调控，RNA结合亲和力并不是PAMP选择的唯一标准。通过一系列独特的纯化蛋白质、RNA试剂，我们将采用互补的生化、生物物理、结构和细胞方法来1)了解RIG-I对RNA的PAMP和非PAMP识别的基础；2)表征RIG-I ATPase的活性及其在RIG-I激活中的作用；以及3)了解RIG-I信号的机制。其结果是更好地理解自我与非自我识别和RIG-I逃避机制，这可能导致广谱抗病毒药物、抗炎治疗药物和基于RNA的基因沉默药物的开发。

DESCRIPTION (provided by applicant): The innate immune system is the first line of defense against microbial and viral infections. A failure to elicit the early innate immune response leads to systemic infections. A significant number of human viruses, including Influenza, Hepatitis C, Dengue, West Nile, Respiratory Syncytial, Reovirus, and Ebola are recognized by the innate immunity receptor RIG-I. The overall goal of this proposal is to understand how RIG-I (Retinoic Acid-inducible Gene-I), a cytoplasmic receptor discriminates normal, cellular from viral RNAs to stimulate a host response. A major goal of our collaborative research studies has been to understand the thermodynamic, kinetic, and structural mechanisms by which RIG-I recognizes atypical pathogen associated molecular pattern (PAMP) feature in RNAs. Our crystal structure of RIG-I bound to blunt-ended dsRNA and an ATP analog established a new paradigm for RIG-I activation. The goal of this proposal is to rigorously test the RIG-I activation model using carefully designed biochemical, kinetic, and structural studies. Our preliminary results demonstrate that RIG-I is regulated in multiple ways and RNA binding affinity is not the only criterion for PAMP selection. With a unique collection of purified protein, RNA reagents, we will employ complementary biochemical, biophysical, structural, and cell based approaches to 1) understand the basis of PAMP versus non-PAMP recognition of RNA by RIG-I; 2) characterize RIG-I ATPase activity and its role in RIG-I activation; and 3) understand the mechanism of RIG-I signaling. The outcomes are better understanding of self versus non-self recognition and RIG-I evasion mechanism, which can lead to the development of broad-spectrum antivirals, anti-inflammatory therapeutics and RNA-based gene silencing agents.