A multipronged investigation of SARS-CoV-2 genome packaging

SARS-CoV-2 基因组包装的多管齐下研究

• 批准号: 10444410
• 负责人: Andrea Soranno
• 金额: $ 63.19万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 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/10444410
• 关键词: 2019-nCoV; Acute; Address; Affinity; Antiviral Agents; Attenuated; Behavior; Binding; Biological Assay; Biophysics; COVID-19 pandemic; Caliber; Cells; Clinical; Coronavirus; Coupled; Data; Development; Disease; Dissection; Distant; 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; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; 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; base; betacoronavirus; coronavirus therapeutics; design; experimental study; genomic RNA; high throughput screening; insight; novel; novel strategies; optical traps; pandemic coronavirus; prevent; screening; simulation; single molecule; small molecule; targeted treatment; zoonotic coronavirus

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.

项目概要 由 SARS-CoV-2 病毒引起的 COVID-19 大流行对人类生命构成了严重且持续的威胁。对病毒生命周期的详细分子了解对于阐明可用于治疗干预的临床可及过程是必要的。核衣壳 (N) 蛋白是一种 420 个残基的多结构域蛋白，具有折叠和无序区域，是基因组包装的基础，是病毒粒子生命周期的重要步骤。 N 蛋白通过结合并压缩基因组 RNA 介导细胞质基因组包装，这一过程在冠状病毒科中显然是保守的。由于缺乏对这些过程的分子理解，我们破坏基因组包装的能力受到限制。为了解决这一知识差距，我们的建议重点关注 N 蛋白如何驱动基因组压缩的分子生物物理学。 N蛋白是高度多价的；它可以通过许多不同的相互作用位点同时结合自身和 RNA。多价在折叠域和本质上无序的区域中编码。尽管对其他冠状病毒的折叠结构域进行了大量研究，但无序区域的分子生物物理学在很大程度上被忽视。我们假设 N 蛋白多价性是 RNA 压缩的分子基础，并且三个无序区域在确定多价性、结合亲和力和 RNA 结合特异性方面发挥着关键作用。通过结合单分子荧光和力谱、系综方法和全原子模拟，我们将剖析这些相互作用背后的分子细节。我们还提出了一种新的小分子筛选方法，该方法利用相分离的蛋白质：RNA 液滴的形成作为基因组压缩的读数。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，并揭示可以减弱基因组压缩的小分子。更一般地说，通过关注从经验上解释其他遥远冠状病毒行为的基本生物物理现象，我们相信我们的结论将广泛适用于代表主要公共卫生威胁的现有冠状病毒（例如SARS、MERS），也适用于未来的新型人畜共患冠状病毒。