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.

蛋白质合成的精确控制对于维持正常细胞功能是必不可少的，并且是细胞内先天性抗病毒反应的核心。例如，蛋白质2 '-5'-寡腺苷酸合成酶（OAS）蛋白家族检测胞质双链（ds）RNA，并通过激活潜在核糖核酸酶L（RNase L）启动翻译控制反应，以限制病毒蛋白质合成并从而复制。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。对于OAS 3，各个结构域结构表明激活过程的关键元件是相似的，但在功能上分为其三个OAS结构域，其中一个（催化失活）结构域主要决定双链RNA结合，另一个远端（活性）OAS结构域负责2 '-5'-寡聚腺苷酸合成。然而，我们发表的关于OAS 1激活RNA序列和结构基序的研究，以及本文提出的大量新的初步数据，强烈认为我们仍然不完全理解特定的RNA特征及其背景如何联合收割机驱动OAS 1和OAS 3的有效激活。特别是，我们未发表的研究表明，OAS 3激活的程度取决于我们为OAS 1定义的一些相同RNA分子特征（序列和结构基序）的作用。该提案继续了我们的创新，多学科研究，重点是定义负责驱动OAS 1和OAS 3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物化学和基于人类细胞的测定，结合生物物理和结构方法，破译控制OAS 1激活的“规则”。具体来说，我们将定义：1）常见RNA结构基序的放置如何影响OAS 1激活RNA标签的功能; 2）能够强烈增强OAS 1激活的dsRNA末端基序的分子作用机制; 3）OAS 1寡聚化在dsRNA上对完全酶激活的作用;以及4）SARS-CoV-2 5 '-UTR激活OAS 1的基础和破坏OAS/RNase L途径对病毒繁殖的影响。在目标2中，我们将使用高分辨率单颗粒冷冻EM来确定与两个细胞非编码RNA结合的全长OAS 3的结构。这些研究将通过以下方式揭示OAS 3激活的分子基础：1）大于公认的50个碱基对（bp）最小长度的确定dsRNA区域，以及2）小于50 bp但含有独特RNA三级基序的RNA，我们假设该基序驱动OAS 3激活的独特模式。这些研究也将为开始定义OAS 3对特定RNA序列和结构基序的依赖性提供一个启动点，就像我们对OAS 1所做的那样。总的来说，这些研究将大大加深我们对RNA调控OAS/RNase L通路的基本理解，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。