dsRNA regulation of the cytosolic innate immune system

胞质先天免疫系统的 dsRNA 调节

• 批准号: 10359208
• 负责人: Graeme L Conn
• 金额: $ 39万
• 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 2023-05-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359208
• 关键词: Active Sites; Adhesions; Adopted; Antiviral Agents; Antiviral Response; Automobile Driving; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biology; Biophysics; Catalysis; Cell Adhesion; Cell Proliferation; Cell physiology; Cells; Characteristics; Complex; Consensus; Coupled; Data; Detection; Development; Double-Stranded RNA; Endoribonucleases; Enzyme Activation; Foundations; Human; Human Pathology; Immune; In Vitro; Infection; Influenza A virus; Innate Immune Response; Innate Immune System; Investigation; Kinetics; Knowledge; Lead; Length; Ligase; Link; Maintenance; Masks; Mediating; Messenger RNA; Modeling; Molecular; Mutagenesis; Natural Immunity; Neoplasm Metastasis; Pathway interactions; Pattern; Phosphotransferases; Polymers; Protein Biosynthesis; Proteins; Proteomics; RNA; Regulation; Research Design; Resistance; Ribonucleases; Role; Route; Second Messenger Systems; Site; Structure; System; Testing; Therapeutic; Transcript; Untranslated RNA; Up-Regulation; Variant; Viral; Viral Proteins; Virus; Virus Diseases; Work; antagonist; base; human disease; innovation; insight; multidisciplinary; novel; novel therapeutics; oligoadenylate; pathogen; pathogenic virus; prevent; response; treatment strategy; 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 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.

蛋白质合成的精确控制对于维持正常细胞功能至关重要，并且对细胞内的先天抗病毒反应至关重要。例如，先天免疫系统蛋白2'-5'-5'-寡胶质酸合成酶（OAS）通过激活潜在的核糖核酸酶L（RNase L）来检测胞质双链（DS）RNA，从而启动转化控制反应，从而限制病毒蛋白质的合成和重复。 OAS1和OAS1-DSRNA复合物的结构揭示了对OAS1激活的重要见解：DSRNA结合驱动了OAS1活性位点的功能必不可少的重组。然而，我们最近发现了一种新型的单链RNA基序，该基序强烈潜在地激活OAS1的激活以及此处介绍的广泛初步数据，强烈认为，我们仍然对特定RNA特征及其上下文的理解仍然有限，以驱动OAS1的潜在激活。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的OAS1结合位点，目前在溶液中直接定义的RNA特征（S）直接结合方向，因此控制了OAS1激活的效力。此外，我们表明人类非编码RNA 886（NC886）包含一种新型的RNA三级结构，是OAS1的独特且具有巨大潜在的激活剂。该提案描述了一项创新的多学科研究，其特定重点是定义负责驱动OAS1激活的RNA特征和环境及其对细胞抗病毒反应的影响。在AIM 1中，我们将使用模型dsRNA的序列和长度变体来破译由dsRNA激活OAS1激活的“规则”。使用体外生物化学和人类细胞基于生物物理，蛋白质组学和结构方法，我们将确定特定的RNA特征在协调或竞争中如何起作用，以驱动OAS1-DSRNA相互作用以及OAS1活性的程度。互补的病毒学测定法将在适当的生物学环境中对DSRNA介导的OAS/RNase L途径的调节进行新的理解，从而对病毒感染进行抗性。在AIM 2中，我们将确定导致其潜在OAS1激活的NC886的分子特征。此外，我们将测试我们的新假设，即在影响力在Atractza A期间的上调通过与病毒NS1蛋白的相互作用特异性地抵消了感染。总的来说，这些研究将通过OAS/RNase L途径揭示对RNA介导的翻译控制的新见解，该途径可以作为定义天然OAS1激活剂（如NC886）和Divers Viruse采用的OAS1进化策略的生物学作用的框架。知识将是开发通常适用的抗病毒治疗方法的基础，并可以通过激活OAS/RNase L途径作为控制转移的增殖/粘附特征的新途径来为治疗其他人类疾病的治疗策略提供信息。