Double-stranded RNA dictates SARS-CoV-2 nucleocapsid condensation temperature

双链 RNA 决定 SARS-CoV-2 核衣壳凝结温度

• 批准号: 10740350
• 负责人: Christine Anne Roden
• 金额: $ 10万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-18 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740350
• 关键词: 2019-nCoV; Address; Biochemical; Biochemistry; Biological Assay; Biological Models; Biological Process; Biology; Biomedical Engineering; COVID-19 treatment; Cells; Cellular Assay; Cellular biology; Chemicals; Collaborations; Coronavirus; Cytoplasm; Development; Dimerization; Disease; Disease Outbreaks; Double-Stranded RNA; Failure; Generations; Genome; Goals; Growth; Health Benefit; Inosine Diphosphate; Lead; Learning; Maps; Membrane; Modeling; Molecular; Molecular Biology; Mutation; Nucleocapsid; Nucleocapsid Proteins; Peptides; Phase; Phenotype; Physical condensation; Physiological; Process; Property; Proteins; Proteome; Public Health; RNA; RNA Binding; RNA Probes; RNA Sequences; RNA Viruses; RNA-Binding Proteins; RNA-Protein Interaction; Research; Research Personnel; Role; Running; Safety; Sensitivity and Specificity; Specific qualifier value; Structural Protein; Structure; System; Techniques; Temperature; Testing; Time; Training; Translational Regulation; Translations; Universities; Viral; Viral Physiology; Virus; Virus Replication; Work; betacoronavirus; biosafety level 3 facility; crosslink; design; dimer; future outbreak; genetic manipulation; inhibitor; interest; novel; novel therapeutics; protein crosslink; protein phosphatase inhibitor-2; recruit; skills; small molecule; small molecule inhibitor; small molecule libraries; symposium; viral RNA; virology

Project Summary/Abstract: INTRODUCTION: RNA viruses such as SARS-CoV-2 represent an ongoing public health threat. To replicate properly, RNA viruses must perform multiple functions in the same cytoplasm (i.e., genome replication, circularization, generation of sub-genomic RNA, packaging etc.) and for betacoronaviruses like SARS-CoV-2 the structural nucleocapsid protein (N) is required for all of these processes. How are RNA viruses able to perform multiple functions in the same cytoplasm with their limited proteome (for example ~24 proteins for SARS-CoV-2)? I hypothesize that one way RNA viruses achieve biochemical complexity is by condensation of RNA and protein components. Biochemical complexity can emerge by virtue of the co-condensing RNA as different viral RNA sequences confer different condensate properties. In this way, a single protein, N, can perform multiple functions in the same cytoplasm. I uncovered the mechanism by which SARS-CoV-2 N recognizes RNA to undergo condensation. Now, I am interested in understanding how the interaction of N with viral RNA controls condensation and impacts important viral functions. RESEARCH: In my K99/R00 research; (Aim 1) I will unbiasedly probe RNA-RNA interactions to understand how RNA is arranged and how interactions are modulated by condensation for genome circularization. (Aim 2) I will develop BSL2 assays to determine which viral processes require N condensation. (Aim 3) I will design synthetic RNA-binding proteins and small molecules to disrupt N condensation. TRAINING: I will complete my training period in Dr. Amy Gladfelter’s lab. During the training period, I will further develop my skills in RNA structure probing SHAPE (RNA structure), RNP-map (RNA/protein crosslinking), and SHAPE-Jump (RNA-RNA crosslinking) in collaboration with Weeks lab. I will learn how to design synthetic IDPs for the creation of bespoke RNA-binding proteins (Chilkoti). I will screen an existing small molecule library to identify inhibitors of N condensation (Fiorti). I will learn how to culture and genetically manipulate a BSL2 model betacoronavirus, MHV (Sheahan and Cameron). ENVIRONMENT: Dr. Amy Gladfelter is one of the world’s leading experts on the role of RNA in condensation. Additionally, UNC has some of the world’s leading experts in coronavirus biology including labs with BSL3 facilities offering the possibility to test newly developed N condensation inhibitors with live virus. I plan to further my growth through participation in RNA and condensation conferences, and attendance of courses in virology. I also plan on using my K99/R00 to ease my transition into running my own lab as an independent investigator. I am particularly interested in joining a university with strong biochemistry, molecular biology, virology, and biomedical engineering departments who are looking for an RNA structure enthusiast to join their ranks! IMPACT ON PUBLIC HEALTH: This work will advance our understanding of how betacoronavirus N recognize viral RNA sequences and may one day lead to novel therapeutics.

项目摘要/摘要： 简介：SARS-CoV-2 等 RNA 病毒是一种持续的公共卫生威胁。复制 正确地说，RNA病毒必须在同一细胞质中执行多种功能（即基因组复制、 环化、亚基因组 RNA 的生成、包装等）以及 SARS-CoV-2 等 β 冠状病毒 所有这些过程都需要结构核衣壳蛋白 (N)。 RNA病毒如何能够 利用其有限的蛋白质组（例如约 24 种蛋白质）在同一细胞质中执行多种功能 SARS-CoV-2）？我假设 RNA 病毒实现生化复杂性的一种方式是通过凝结 RNA 和蛋白质成分。生化复杂性可以通过共缩合 RNA 出现： 不同的病毒RNA序列赋予不同的凝聚物特性。这样，单个蛋白质 N 就可以 在同一细胞质中执行多种功能。我发现了 SARS-CoV-2 N 的机制 识别RNA进行缩合。现在，我有兴趣了解 N 与 病毒 RNA 控制凝结并影响重要的病毒功能。研究：在我的 K99/R00 研究中； （目标 1）我将公正地探究 RNA-RNA 相互作用，以了解 RNA 是如何排列的以及如何 相互作用通过基因组环化的缩合来调节。 （目标 2）我将开发 BSL2 检测方法 确定哪些病毒过程需要氮气凝结。 （目标 3）我将设计合成的 RNA 结合蛋白 和小分子来破坏 N 凝结。培训：我将在艾米博士那里完成我的培训期 格拉德费尔特的实验室。在培训期间，我将进一步发展我在RNA结构探测SHAPE方面的技能 （RNA 结构）、RNP-map（RNA/蛋白质交联）和 SHAPE-Jump（RNA-RNA 交联） 与 Weeks 实验室合作。我将学习如何设计合成 IDP 以创建定制的 RNA 结合 蛋白质（奇尔科蒂）。我将筛选现有的小分子库来识别 N 缩合抑制剂 （菲奥尔蒂）。我将学习如何培养和基因操作 BSL2 模型 β 冠状病毒，MHV（Sheahan 和卡梅伦）。环境：Amy Gladfelter 博士是研究 RNA 在生物体内的作用方面的世界领先专家之一。 缩合。此外，北卡罗来纳大学拥有一些世界领先的冠状病毒生物学专家，包括实验室 BSL3 设施提供了用活病毒测试新开发的 N 缩合抑制剂的可能性。我 计划通过参加 RNA 和凝结会议以及出席 病毒学课程。我还计划使用 K99/R00 来轻松过渡到运行我自己的实验室 独立调查员。我特别有兴趣加入一所生物化学、分子学科较强的大学 正在寻找 RNA 结构爱好者的生物学、病毒学和生物医学工程系 加入他们的行列！对公众健康的影响：这项工作将增进我们对如何 β冠状病毒 N 识别病毒 RNA 序列，有一天可能会带来新的治疗方法。