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。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，我们发表的关于OAS1激活RNA序列和结构基序的研究，以及这里提供的大量新的初步数据，强烈认为我们仍然不完全了解特定的RNA特征及其上下文如何结合来驱动OAS1和OAS3的有效激活。特别是，我们未发表的研究表明，OAS3的激活程度取决于我们为OAS1定义的一些相同的RNA分子签名(序列和结构基序)的作用。这项建议继续了我们的创新的多学科研究，特别侧重于确定负责驱动OAS1和OAS3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，我们将使用体外生化和基于人体细胞的分析，结合生物物理和结构方法，继续破译管理OAS1激活的“规则”。在目标2中，我们将使用高分辨率单粒子冷冻EM来确定与两个细胞非编码RNA结合的全长OAS3的结构。这些研究将通过以下两个方面揭示OAS3激活的分子基础：1)定义的dsRNA区域大于公认的50个碱基对(BP)的最小长度；2)小于50bp但包含独特的RNA第三基序的RNA，我们假设该基序驱动OAS3激活的一种独特模式。这些研究也将提供一个起点，开始定义OAS3的S依赖于特定的RNA序列和结构基序的有效激活，就像我们对OAS1所做的那样。综上所述，这些研究将大大加深我们对核糖核酸对OAS/核糖核酸酶L通路调控的基本了解，并可能为开发新的、普遍适用的抗病毒治疗方法提供必要的基础。

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 protein 2’-5’-oligoadenylate synthetase (OAS) family of proteins detects cytosolic double-stranded (ds)RNA and initiates a translational control response via activation of the latent ribonuclease L (RNase L), to limit 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. For OAS3, individual domain structures suggest that key elements of the activation process are similar but functionally divided among its three OAS domains, with one (catalytically inactive) domain primarily dictating dsRNA binding and a another distant (active) OAS domain responsible for 2’-5’-oligoadenylate synthesis. However, our published studies on OAS1-activating RNA sequence and structural motifs, and extensive new preliminary data presented here, strongly argue that we still have an incomplete understanding of how specific RNA features and their contexts combine to drive potent activation of OAS1 and OAS3. In particular, our unpublished studies indicate that the extent of OAS3 activation is dependent on the action of some of the same RNA molecular signatures (sequence and structural motifs) that we have defined for OAS1. This proposal continues our innovative, multidisciplinary study with a specific focus on defining the RNA features and contexts responsible for driving OAS1 and OAS3 activation and their resultant impacts on the cellular antiviral response. In Aim 1, we will continue deciphering the “rules” that govern OAS1 activation using in vitro biochemical and human cell-based assays, coupled with biophysical and structural approaches. Specifically, we will define: 1) how placement of common RNA structural motifs affects the function of OAS1-activating RNA signatures; 2) the molecular mechanism of action of dsRNA terminal motifs capable of strongly enhancing OAS1 activation; 3) the proposed role for OAS1 oligomerization on dsRNA for full enzyme activation; and 4) the basis of OAS1 activation by the SARS-CoV-2 5’-UTR and the impact of disrupting the OAS/RNase L pathway on viral propagation. In Aim 2, we will use high- resolution single-particle cryo-EM to determine the structures of full-length OAS3 bound to two cellular non- coding RNAs. These studies will reveal the molecular basis for OAS3 activation by 1) a defined dsRNA region larger than the accepted 50 base pair (bp) minimum length, and 2) an RNA with less than 50bp but containing a unique RNA tertiary motif that we hypothesize drives a distinct mode of OAS3 activation. These studies will also provide a launch point to begin defining OAS3’s dependence on specific RNA sequence and structural motifs for its potent activation as we have done for OAS1. Collectively, these studies will substantially deepen our fundamental understanding of OAS/RNase L pathway regulation by RNA and may offer an essential foundation for development of novel, generally applicable anti-viral therapeutic approaches.