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）通过激活潜在核糖核酸酶L（RNase L）来检测胞质双链（ds）RNA以启动翻译控制应答，这限制了病毒蛋白质合成并因此限制了复制。OAS 1和OAS 1-dsRNA复合物的结构揭示了OAS 1激活的重要见解：dsRNA结合驱动OAS 1活性位点的功能性重组。然而，我们最近发现了一种新的单链RNA基序，它强烈增强了OAS 1的激活，以及这里提供的大量初步数据，强烈认为我们对特定RNA特征及其背景如何结合联合收割机来驱动OAS 1的有效激活的理解仍然有限。我们的新数据表明，用于OAS 1结构研究的模型dsRNA包含竞争性激活和非激活OAS 1结合位点，并且目前定义不清的RNA特征指导溶液中的结合方向，从而控制OAS 1激活的效力。此外，我们表明，人类非编码RNA 886（nc 886）包含一个新的RNA三级结构，是一个独特的和非常有效的激活OAS 1。该提案描述了一项创新的多学科研究，重点是定义负责驱动OAS 1激活的RNA特征和背景及其对细胞抗病毒反应的影响。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OAS 1的“规则”。使用体外生物化学和基于人类细胞的测定，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工作，以驱动OAS 1-dsRNA相互作用和OAS 1活性的程度。互补病毒学检测将把这种新的理解dsRNA介导的调节OAS/RNase L途径，从而抵抗病毒感染，在适当的生物学背景。在目标2中，我们将确定导致其有效激活OAS 1的nc 886的分子特征。此外，我们将测试我们的新假设，即在甲型流感感染过程中nc 886的上调是通过与病毒NS 1蛋白的相互作用而特异性抵消的。总的来说，这些研究将揭示通过OAS/RNase L途径对RNA介导的翻译控制的新见解，该途径可作为定义天然OAS 1激活剂（如nc 886）的生物学作用和不同病毒采用的OAS 1逃避策略的框架。这些知识将是开发普遍适用的抗病毒治疗方法的必要基础，并且可以为治疗其他人类疾病的策略提供信息，例如通过激活OAS/RNA酶L途径作为控制转移的增殖/粘附特征的新途径。

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.