A multipronged investigation of SARS-CoV-2 genome packaging

SARS-CoV-2 基因组包装的多管齐下研究

基本信息

批准号：
10610414
负责人：
Andrea Soranno
金额：
$ 62.59万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-16 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10610414
关键词：
2019-nCoV Acute Address Affinity Antiviral Agents Attenuated Behavior Binding Biological Assay Biophysics COVID-19 pandemic Cells Clinical Coronavirus Coupled Data Development Diameter Disease Dissection Distant Encapsulated Family Fluorescence Fluorescence Spectroscopy Future Genome Goals Human In Vitro Investigation Knowledge Lead Length Life Life Cycle Stages Liquid substance Measurement Measures Mediating Methods Microscope Microscopy Middle East Respiratory Syndrome Molecular N Domain Nucleic Acids Nucleocapsid Phase Physical condensation Play Process Proteins Public Health Publishing RNA RNA Binding RNA Viruses RNA-Binding Proteins RNA-Protein Interaction Resolution Role SARS-CoV-2 genome Severe Acute Respiratory Syndrome Site Specificity Spectrum Analysis Tertiary Protein Structure Testing Therapeutic Therapeutic Intervention Vaccines Viral Viral Genome Viral Packaging Virion Virus Virus Replication Work betacoronavirus coronavirus pandemic coronavirus therapeutics design experimental study genomic RNA high throughput screening insight novel novel strategies optic trap optical traps prevent screening simulation single molecule small molecule targeted treatment zoonotic coronavirus

项目摘要

Project Summary The COVID-19 pandemic, caused by the virus SARS-CoV-2, represents an acute and ongoing threat to human life. A detailed molecular understanding of the viral life cycle is necessary to illuminate clinically accessible processes that can be targeted for therapeutic intervention. The Nucleocapsid (N) protein is a 420-residue multidomain protein with both folded and disordered regions that underlies genome packaging, an essential step in the virion lifecycle. N protein mediates cytosolic genome packaging by binding to and compacting genomic RNA in a process apparently conserved across the coronaviridae family. Our ability to disrupt genome packaging is limited by the absence of a molecular understanding of these processes. To address this knowledge gap, our proposal is focused on the molecular biophysics that underlies how N protein drives genome compaction. N protein is highly multivalent; it can simultaneously bind to both itself and RNA via a number of distinct interaction sites. Multivalency is encoded across both folded domains and intrinsically disordered regions. While there has been substantial work on the folded domains in other coronaviruses, the molecular biophysics of the disordered regions has been largely ignored. We hypothesize N protein multivalency underlies the molecular basis of RNA compaction, and that the three disordered regions play key roles in determining multivalency, binding affinity, and RNA binding specificity. Through the combination of single-molecule fluorescence and force spectroscopy, ensemble methods, and all-atom simulation, we will dissect the molecular details that underlie these interactions. We also present a novel approach to small-molecule screening that leverages the formation of phase separated protein:RNA liquid droplets as a readout for genome compaction. Our work will offer high-resolution structural insight into the physical basis for two critical steps in the viral life cycle, as well as reveal small molecules that can attenuate genome compaction. More generally, by focusing on fundamental biophysical phenomena that empirically explain behavior from other distant coronaviruses, we believe that our conclusions will be broadly transferable to existing coronaviruses that represent major public health threats (e.g., SARS, MERS) but also to future novel zoonotic coronaviruses.

项目摘要由病毒SARS-COV-2引起的COVID-19大流行代表了对人类生命的急剧威胁。对病毒生命周期的详细分子理解对于阐明可用于治疗干预的临床上可访问过程的过程是必要的。 Nucleocapsid（N）蛋白是一种420个残留的多域蛋白，具有折叠和无序区域，是基因组包装的基础，这是病毒率生命周期中必不可少的一步。 N蛋白通过在明显保守的冠状病毒家族的过程中与压实基因组RNA结合并压实基因组RNA来介导胞质基因组包装。我们破坏基因组包装的能力受到对这些过程的分子理解的限制。为了解决这一知识差距，我们的建议集中于N蛋白如何驱动基因组压实的分子生物物理学。 N蛋白具有高度多价。它可以通过许多不同的相互作用位点同时与自身和RNA结合。多价均在两个折叠域和内在无序区域进行编码。尽管在其他冠状病毒中的折叠结构域进行了大量工作，但无序区域的分子生物物理学在很大程度上被忽略了。我们假设N蛋白多价构成了RNA压实的分子基础，并且三个无序区域在确定多价，结合亲和力和RNA结合特异性方面起着关键作用。通过单分子荧光和力光谱，集合方法和全原子模拟的组合，我们将剖析这些相互作用的分子细节。我们还提出了一种新型的小分子筛选方法，该方法利用了相分离蛋白的形成：RNA液滴作为基因组压实的读数。我们的工作将为病毒生命周期中两个关键步骤的物理基础提供高分辨率的结构洞察力，并揭示可以减弱基因组压实的小分子。更一般而言，通过关注基本的生物物理现象，从经验上解释了其他遥远的冠状病毒的行为，我们认为我们的结论将广泛转移到代表主要公共卫生威胁（例如SARS，MERS）的现有冠状病毒中，但也可以将未来的新颖性冠状动脉转移。