dsRNA regulation of the cytosolic innate immune system

蛋白质合成的精确控制对于维持细胞的正常功能至关重要，也是细胞内先天抗病毒反应的核心。例如，天然免疫系统蛋白2‘-5’-寡腺苷合成酶(OAS)通过激活潜伏的核糖核酸酶L(核糖核酸酶L)，检测胞质双链(DS)RNA，启动翻译控制反应，从而限制病毒蛋白质的合成和复制。OAS1和OAS1-dsRNA复合体的结构揭示了对OAS1激活的重要见解：dsRNA结合驱动OAS1活性位点的功能重组。然而，我们最近发现了一个新的单链RNA基序，它强烈地增强了OAS1的激活，并且提供了大量的初步数据，我们强烈地认为，我们仍然对特定的RNA特征及其上下文如何结合来驱动OAS1的有效激活有有限的理解。我们的新数据表明，用于OAS1结构研究的模型dsRNA包含竞争激活和非激活OAS1结合位点，目前定义不清的RNA特征(S)指导了溶液中的结合方向，从而控制了OAS1激活的效力。此外，我们发现人类非编码RNA 886(Nc886)包含一种新的RNA三级结构，它是OAS1的一种独特且非常有效的激活剂。这项提案描述了一项创新的多学科研究，重点是确定负责驱动OAS1激活及其对细胞抗病毒反应的影响的RNA特征和背景。在目标1中，我们将使用模型dsRNA的序列和长度变体来破译管理dsRNA激活OAS1的“规则”。使用体外生化和基于人类细胞的分析，结合生物物理学、蛋白质组学和结构方法，我们将确定特定的RNA签名如何协同或竞争地驱动OAS1-dsRNA相互作用和OAS1活性的程度。互补的病毒学分析将把这种对双链RNA介导的OAS/核糖核酸酶L途径的调控，从而对病毒感染的抵抗力的新理解置于适当的生物学背景下。在目标2中，我们将确定导致其对OAS1有效激活的分子特征(S)。此外，我们将检验我们的新假设，即在甲型流感感染期间，nc886的上调是通过与病毒NS1蛋白的相互作用特异性地对抗的。总之，这些研究将揭示通过oas/rna酶L途径对RNA介导的翻译控制的新见解，该途径可能作为一个框架来定义天然oas1激活物(如nc886)的生物学作用(S)以及各种病毒采取的oas1规避策略。这些知识将是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参考，例如通过激活OAS/核糖核酸酶L途径作为一种新的途径来控制转移的增殖/黏附特征。

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.