dsRNA regulation of the cytosolic innate immune system

胞质先天免疫系统的 dsRNA 调节

• 批准号: 10736791
• 负责人: Graeme L Conn
• 金额: $ 46万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-12 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736791
• 关键词: 2-5A Synthetase; 2019-nCoV; Active Sites; Adhesions; Affect; Antiviral Agents; Antiviral Response; Automobile Driving; Award; Base Pairing; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biology; Biophysics; Cell Adhesion; Cell Proliferation; Cell physiology; Cells; Complex; Consensus Sequence; Coupled; Cryoelectron Microscopy; Data; Dependence; Detection; Development; Distant; Double-Stranded RNA; Elements; Endoribonucleases; Enzyme Activation; Foundations; Human; Human Pathology; In Vitro; Individual; Innate Immune Response; Innate Immune System; Length; Ligase; Link; Maintenance; Mediating; Messenger RNA; Modeling; Molecular; Molecular Mechanisms of Action; Molecular Profiling; Pathogen detection; Pathway interactions; Pattern; Polymers; Positioning Attribute; Process; Productivity; Protein Biosynthesis; Protein Family; Proteins; Publishing; RNA; RNA Sequences; Regulation; Resistance; Resolution; Ribonucleases; Role; Second Messenger Systems; Structure; System; Testing; Therapeutic; Untranslated RNA; Untranslated Regions; Viral; Viral Proteins; Virus; Virus Diseases; innovation; insight; interdisciplinary collaboration; multidisciplinary; novel; novel therapeutics; oligoadenylate; particle; pathogen; pathogenic virus; response; tripolyphosphate; viral RNA

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.

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，检测蛋白质胞质双链 (ds) 的蛋白质 2'-5'-寡腺苷酸合成酶 (OAS) 家族。 )RNA 并通过激活潜在核糖核酸酶 L (RNase L) 启动翻译控制反应，以限制病毒蛋白合成，从而限制 OAS1 和结构的复制。 OAS1-dsRNA 复合物揭示了对 OAS1 激活的重要见解：dsRNA 结合驱动 OAS1 活性位点功能上必需的重组，对于 OAS3，各个结构域结构表明激活过程的关键要素相似，但在功能上分为三个 OAS 结构域。一个（催化非活性）结构域主要决定 dsRNA 结合，另一个远程（活性）OAS 结构域负责 2'-5'-寡腺苷酸合成。对 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表明，我们对特定 RNA 特征及其背景如何结合起来驱动 OAS1 和 OAS3 的有效激活的了解仍然不完全。未发表的研究表明，OAS3 激活的程度取决于我们为 OAS1 定义的一些相同 RNA 分子特征（序列和结构基序）的作用。该提案继续我们的创新、多学科研究，特别侧重于定义。负责驱动 OAS1 和 OAS3 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，我们将继续使用体外生化和基于人类细胞的检测来破译控制 OAS1 激活的“规则”。具体而言，我们将定义：1) 常见 RNA 结构基序的放置如何影响 OAS1 激活 RNA 特征的功能；2) 的分子作用机制。能够强烈增强 OAS1 激活的 dsRNA 末端基序；3) dsRNA 上 OAS1 寡聚化对完全酶激活的拟议作用；以及 4) SARS-CoV-2 5'-UTR 激活 OAS1 的基础以及破坏OAS/RNase L 通路对病毒传播的影响 在目标 2 中，我们将使用高分辨率单颗粒冷冻电镜来确定与两个蛋白结合的全长 OAS3 的结构。这些研究将通过以下方式揭示 OAS3 激活的分子基础：1）大于公认的 50 碱基对 (bp) 最小长度的明确 dsRNA 区域，以及 2) 小于 50bp 但包含独特 RNA 的 RNA。我们开创的第三基序驱动 OAS3 激活的独特模式，这些研究还将提供一个起点，以开始定义 OAS3 对其有效激活的特定 RNA 序列和结构基序的依赖，正如我们所做的那样。总的来说，这些研究将大大加深我们对 RNA 调节 OAS/RNase L 通路的基本理解，并可能为开发新型、普遍适用的抗病毒治疗方法提供重要基础。