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.

项目概要/摘要： RNA病毒如SARS-CoV-2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的产生、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病毒是如何 在相同的细胞质中以其有限的蛋白质组执行多种功能（例如， SARS-CoV-2）？我假设RNA病毒获得生物化学复杂性的一种方式是通过浓缩 RNA和蛋白质成分。生物化学的复杂性可以通过RNA的共凝聚而出现， 不同的病毒RNA序列赋予不同的缩合物性质。通过这种方式，单个蛋白质N可以 在同一细胞质中执行多种功能。我发现了SARS-CoV-2N 识别RNA进行缩合。现在，我感兴趣的是了解N与 病毒RNA控制浓缩并影响重要的病毒功能。研究：在我的K99/R 00研究中; (Aim 1）我将无偏见地探测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。(Aim 2）我将开发BSL 2检测试剂盒， 确定哪些病毒过程需要N缩合。(Aim 3）设计合成的RNA结合蛋白 和小分子来破坏N缩合。培训：我将在艾米博士完成我的培训期 格拉菲尔特的实验室。在培训期间，我将进一步发展我的RNA结构探测SHAPE技能 (RNA结构）、RNP-映射（RNA/蛋白质交联）和SHAPE-跳跃（RNA-RNA交联）， 与Weeks实验室合作。我将学习如何设计合成的IDPs，以创建定制的RNA结合 蛋白质（Chilkoti）。我将筛选一个现有的小分子库，以确定N缩合抑制剂 （Fiorti）.我将学习如何培养和遗传操作BSL 2模型β冠状病毒，MHV（Sheahan 和卡梅隆）。环境：Amy Gladfelter博士是世界上研究RNA作用的主要专家之一， 凝此外，该公司拥有一些世界领先的冠状病毒生物学专家，包括实验室 BSL 3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 我计划通过参加RNA和缩合会议，以及参加 病毒学课程我还计划使用我的K99/R 00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、分子生物学 生物学，病毒学和生物医学工程部门谁正在寻找一个RNA结构爱好者， 加入他们的行列！对公众健康的影响：这项工作将促进我们对如何 β冠状病毒N识别病毒RNA序列，有一天可能导致新的治疗方法。