A multipronged investigation of SARS-CoV-2 genome packaging

项目摘要 由SARS-CoV-2病毒引起的新冠肺炎大流行对人类生命构成了严重和持续的威胁。对病毒生命周期的详细分子理解是必要的，以阐明可以作为治疗干预目标的临床可获得的过程。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关键步骤。N蛋白通过与基因组RNA结合并压缩基因组RNA来介导细胞质基因组包装，这一过程显然在冠状病毒科家族中是保守的。我们扰乱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，我们的建议集中在N蛋白如何驱动基因组紧凑的分子生物物理学上。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。多价性是跨折叠结构域和本质无序区域编码的。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在很大程度上被忽视了。我们假设N蛋白的多价性是RNA紧凑的分子基础，并且这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，该方法利用相分离蛋白质的形成：RNA液滴作为基因组压缩的读数。我们的工作将提供对病毒生命周期两个关键步骤的物理基础的高分辨率结构洞察，以及揭示可以减弱基因组紧凑的小分子。更广泛地说，通过专注于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)，以及未来新型人畜共患病冠状病毒。

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.