dsRNA regulation of the cytosolic innate immune system

精确控制蛋白质合成对于维持正常细胞功能至关重要，并且是细胞内先天抗病毒反应的核心。例如，先天免疫系统蛋白2'-5'-寡腺苷酸合成酶（OAS）通过激活潜伏核糖核酸酶L （RNase L）来检测胞质双链RNA，从而启动翻译控制反应，从而限制病毒蛋白的合成和复制。OAS1和OAS1-dsRNA复合物的结构揭示了OAS1激活的重要见解：dsRNA结合驱动OAS1活性位点的功能基本重组。然而，我们最近发现了一种新的单链RNA基序，它强烈地增强了OAS1的激活，以及本文提供的大量初步数据，强烈地表明我们对特定RNA特征及其背景如何结合起来驱动OAS1的有效激活的理解仍然有限。我们的新数据表明，用于OAS1结构研究的模型dsRNA包含竞争性的激活和非激活OAS1结合位点，并且目前不明确的RNA在溶液中具有直接结合取向，从而控制OAS1激活的有效性。此外，我们发现人类非编码RNA 886 （nc886）含有一种新的RNA三级结构，是一种独特且非常有效的OAS1激活剂。该提案描述了一项创新的多学科研究，特别关注定义负责驱动OAS1激活的RNA特征和背景及其对细胞抗病毒反应的最终影响。在目标1中，我们将使用模型dsRNA的序列和长度变体来破译控制dsRNA激活OAS1的“规则”。利用体外生化和基于人类细胞的分析，结合生物物理、蛋白质组学和结构方法，我们将确定特定RNA特征如何协同或竞争地工作，以驱动OAS1- dsrna相互作用和OAS1活性的程度。补充病毒学分析将在适当的生物学背景下对dsrna介导的OAS/RNase L途径的调节以及对病毒感染的抗性进行新的理解。在Aim 2中，我们将确定nc886导致其有效激活OAS1的分子特征。此外，我们将验证我们的新假设，即甲型流感感染期间nc886的上调通过与病毒NS1蛋白的相互作用被特异性地抵消。总的来说，这些研究将揭示通过OAS/RNase L途径介导的rna介导的翻译控制的新见解，这可能作为定义天然OAS1激活剂（如nc886）的生物学作用和各种病毒采用的OAS1逃避策略的框架。这些知识将成为开发普遍适用的抗病毒治疗方法的重要基础，并可以为其他人类疾病的治疗策略提供信息，例如，通过激活OAS/RNase L途径作为控制转移的增殖/粘附特性的新途径。

Precise control of protein synthesis is essential for maintenance of normal cellular function and is central to innate antiviral responses within the cell. For example, the innate immune system protein 2'-5'-oligoadenylate synthetase (OAS) detects cytosolic double-stranded (ds)RNA to initiate a translational control response, via activation of the latent ribonuclease L (RNase L), which limits viral protein synthesis and thus replication. Structures of OAS1 and OAS1-dsRNA complexes have revealed important insights into OAS1 activation: dsRNA binding drives a functionally essential reorganization of the OAS1 active site. However, our recent discovery of a novel single-stranded RNA motif which strongly potentiates activation of OAS1, and extensive preliminary data presented here, strongly argue that we still have limited understanding of how specific RNA features and their contexts combine to drive potent activation of OAS1. Our new data show that the model dsRNA used for OAS1 structural studies contains competing activating and non-activating OAS1 binding sites, and that currently ill-defined RNA feature(s) direct binding orientation in solution and thus control the potency of OAS1 activation. Further, we show that the human non-coding RNA 886 (nc886) contains a novel RNA tertiary structure that is a unique and remarkably potent activator of OAS1. This proposal describes an innovative, multidisciplinary study with a specific focus on defining the RNA features and contexts responsible for driving OAS1 activation and their resultant impacts on the cellular antiviral response. In Aim 1, we will use sequence and length variants of a model dsRNA to decipher the “rules” that govern OAS1 activation by dsRNA. Using in vitro biochemical and human cell-based assays coupled with biophysical, proteomic, and structural approaches, we will determine how specific RNA signatures work, cooperatively or in competition, to drive OAS1-dsRNA interaction and the extent of OAS1 activity. Complementary virological assays will place this new understanding of dsRNA-mediated regulation of the OAS/RNase L pathway, and thus resistance to viral infection, in an appropriate biological context. In Aim 2, we will determine the molecular feature(s) of nc886 that lead to its potent activation of OAS1. Further, we will test our novel hypothesis that upregulation of nc886 during influenza A infection is specifically countered by interaction with the viral NS1 protein. Collectively, these studies will reveal novel insights into RNA-mediated translational control via the OAS/RNase L pathway that may serve as a framework to define the biological role(s) of natural OAS1 activators such as nc886 and the OAS1 evasion strategies adopted by diverse viruses. Such knowledge will be an essential foundation for development of generally applicable anti-viral therapeutic approaches and can inform strategies for treatment of other human diseases, for example by activating the OAS/RNase L pathway as a novel route to control the proliferation/ adhesion characteristic of metastasis.