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.

项目摘要 由SARS-CoV-2病毒引起的COVID-19大流行对人类生命构成严重和持续的威胁。病毒生命周期的详细分子理解是必要的，以阐明临床上可访问的过程，可以有针对性的治疗干预。核衣壳（N）蛋白是具有折叠和无序区域的420个残基的多结构域蛋白，其是基因组包装的基础，基因组包装是病毒体生命周期中的重要步骤。N蛋白介导的细胞溶质基因组包装结合和压缩基因组RNA的过程中，显然保守的冠状病毒科。我们破坏基因组包装的能力受到缺乏对这些过程的分子理解的限制。为了解决这一知识差距，我们的建议是集中在分子生物物理学的基础上如何N蛋白驱动基因组压缩。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与自身和RNA结合。多价性在折叠结构域和内在无序区域中编码。虽然在其他冠状病毒的折叠结构域上已经有了大量的工作，但无序区域的分子生物物理学在很大程度上被忽视了。我们假设N蛋白的多价性是RNA压缩的分子基础的基础，并且三个无序区域在决定多价性、结合亲和力和RNA结合特异性方面发挥关键作用。通过单分子荧光和力谱，系综方法和全原子模拟的结合，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见解，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转移到代表主要公共卫生威胁的现有冠状病毒（例如，SARS，MERS），而且还涉及未来的新型人畜共患冠状病毒。