SARS-CoV-2 N interactions with RNA and host cell cyclophilin-A

SARS-CoV-2 N 与 RNA 和宿主细胞亲环蛋白-A 的相互作用

• 批准号: 10622478
• 负责人: ELAN Z EISENMESSER
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622478
• 关键词: 2019-nCoV; Active Sites; Address; Affinity; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; Cells; Collaborations; Complement; Complex; Coronavirus; Coronavirus nucleocapsid protein; Cyclophilin A; Cyclophilins; Detection; Development; Dimerization; Disease; Foundations; Human; Infection; Life Cycle Stages; Membrane; Methods; Middle East Respiratory Syndrome; Molecular; N-terminal; Nature; Nucleocapsid; Nucleocapsid Proteins; Pathogenicity; Physiological; Population; Protein Dynamics; Proteins; RNA; RNA Binding; Relaxation; Resolution; Role; SARS coronavirus; Site; Structural Protein; Temperature; Therapeutic; Vaccines; Viral; Viral Genome; Viral Proteins; Virulent; biophysical techniques; experience; genomic RNA; human pathogen; inhibitor; macromolecule; pathogen

PROJECT SUMMARY Coronaviruses (CoVs) have emerged as formidable human pathogens since their detection within human populations only 50 years ago, underlying the need to rapidly characterize their molecular mechanisms in order to thwart their infections through therapeutics. Seven CoVs that infect humans are known with a range of pathogenicity, which include the more recent virulent CoVs such as MERS, SARS-CoV-1 and SARS-CoV-2. CoVs comprise four structural proteins that includes the envelope (E), membrane (M), spike (S), and nucleocapsid (N) proteins. CoV N proteins perform numerous functions during the viral life-cycle that includes both packaging genomic RNA and manipulating the host cell machinery, making the N protein the most abundantly expressed viral protein during infection. However, the unique and diverse functions of CoV N proteins have made standard structural methods that address its molecular interactions difficult. For example, such difficulties include their promiscuity in RNA binding, engaging multiple binding sites simultaneously, and the presence of inherently dynamic regions thought to be critical for engaging RNA and host proteins. NMR offers a solution to such challenges, as multiple binding modes can be simultaneously characterized both dynamically and structurally and we have previously shown that the inherently dynamic regions within a CoV N protein (SARS-CoV-1) can be studied by NMR. Thus, the exploratory nature of this R21 proposal is to develop strategies aimed at elucidating the molecular details that underlie the SARS-CoV-2 N protein interactions with RNA (Aim 1) and host proteins (Aim 2). Based on our preliminary studies, we hypothesize that specific regions within the N protein have preferred RNA binding sites (Aim 1) and host cell cyclophilin-A binding sites (Aim 2). We will address these aims through the following: Aim 1) Determine how the SARS-CoV-2 N protein targets RNA. Biochemical and biophysical approaches that include NMR will be used to identify the high affinity binding sites of the N protein within sequences derived from genomic RNA and identify the associated dynamic and structural changes that occur upon complex formation. Aim 2) Determine how the SARS-CoV-2 N protein targets host cell cyclophilin-A. NMR will be used to determine whether the N protein targets the cyclophilin-A active site and whether the N protein is a substrate.

项目概要 自从在体内被发现以来，冠状病毒（CoV）已成为可怕的人类病原体。 人类群体仅在 50 年前出现，因此需要快速表征其分子机制 以通过治疗阻止他们的感染。已知有七种感染人类的​​冠状病毒 致病性，其中包括最近的毒力 CoV，如 MERS、SARS-CoV-1 和 SARS-CoV-2。 冠状病毒由四种结构蛋白组成，包括包膜 (E)、膜 (M)、刺突 (S) 和 核衣壳 (N) 蛋白。 CoV N 蛋白在病毒生命周期中发挥多种功能，包括 既包装基因组RNA又操纵宿主细胞机器，使得N蛋白成为最重要的 感染期间大量表达病毒蛋白。然而，冠状病毒N独特且多样化的功能 蛋白质使得解决其分子相互作用的标准结构方法变得困难。例如， 这些困难包括它们在RNA结合方面的混杂性、同时接合多个结合位点，以及 固有动态区域的存在被认为对于 RNA 和宿主蛋白的结合至关重要。核磁共振 为此类挑战提供了解决方案，因为可以同时表征多种结合模式 动态和结构上，我们之前已经表明，冠状病毒 N 内固有的动态区域 蛋白质 (SARS-CoV-1) 可以通过 NMR 进行研究。因此，这个 R21 提案的探索性本质是开发 旨在阐明 SARS-CoV-2 N 蛋白相互作用的分子细节的策略 RNA（目标 1）和宿主蛋白（目标 2）。 根据我们的初步研究，我们假设 N 蛋白内的特定区域具有 优选的 RNA 结合位点（目标 1）和宿主细胞亲环蛋白-A 结合位点（目标 2）。我们将解决这些 通过以下目标： 目标 1) 确定 SARS-CoV-2 N 蛋白如何靶向 RNA。生物化学和生物物理方法 包括 NMR 在内的技术将用于识别序列中 N 蛋白的高亲和力结合位点 从基因组 RNA 中提取并识别发生的相关动态和结构变化 复杂的形成。 目标 2) 确定 SARS-CoV-2 N 蛋白如何靶向宿主细胞亲环蛋白-A。 NMR 将用于 确定 N 蛋白是否靶向亲环蛋白-A 活性位点以及 N 蛋白是否为底物。