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

项目摘要/摘要： 前言：像SARS-CoV-2这样的RNA病毒是一个持续的公共健康威胁。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)对于像SARS-CoV-2这样的贝塔冠状病毒 结构核衣壳蛋白(N)是所有这些过程所必需的。RNA病毒是如何能够 利用其有限的蛋白质组在同一细胞质中执行多种功能(例如，~24个蛋白质用于 SARS-CoV-2)？我假设，RNA病毒达到生化复杂性的一种方式是通过凝聚 核糖核酸和蛋白质成分。生物化学的复杂性可以通过共凝聚的RNA作为 不同的病毒RNA序列具有不同的凝集物特性。通过这种方式，单一的蛋白质N可以 在同一细胞质中执行多种功能。我揭示了SARS-CoV-2N 识别RNA发生缩合。现在，我感兴趣的是了解N和N的相互作用 病毒RNA控制凝聚并影响重要的病毒功能。研究：在我的K99/R00研究中； (目标1)我将不偏不倚地探索RNA-RNA的相互作用，以了解RNA是如何排列的以及如何 相互作用由基因组循环的缩合来调节。(目标2)我将开发BSL2检测方法以 确定哪些病毒过程需要N冷凝。(目标3)我将设计合成的RNA结合蛋白 和小分子来破坏N的缩合。培训：我将在艾米医生那里完成我的培训 格拉菲特的实验室。在培训期间，我将进一步发展我的RNA结构探测形状的技能 (RNA结构)、RNP-MAP(RNA/蛋白质交联)和SHAPE-Jump(RNA-RNA交联) 与Week实验室合作。我将学习如何设计合成的IDP来创建定制的RNA结合 蛋白质(奇尔科蒂)。我将筛选现有的小分子文库以确定N缩合的抑制剂 (菲奥蒂)我将学习如何培养和遗传操作BSL2模型的BetacoronaVirus，MHV(Sheahan 和卡梅伦)。环境：艾米·格拉菲尔特博士是研究核糖核酸在人类基因组中的作用的世界领先专家之一 冷凝。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的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和缩写会议，以及参加 病毒学课程。我还计划使用我的K99/R00来轻松过渡到以 独立调查员。我特别感兴趣的是加入一所生物化学、分子化学很强的大学 生物学、病毒学和生物医学工程系正在寻找RNA结构爱好者 加入他们的行列吧！对公众健康的影响：这项工作将增进我们对 贝塔冠状病毒N识别病毒RNA序列，有朝一日可能会导致新的治疗方法。

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.