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: 10349084
• 负责人: ELAN Z EISENMESSER
• 金额: $ 19.44万
• 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 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10349084
• 关键词: 2019-nCoV; Active Sites; Address; Affinity; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; COVID-19; 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; Nature; Nucleocapsid; Nucleocapsid Proteins; Pathogenicity; Physiological; Population; Proteins; RNA; RNA Binding; Relaxation; Resolution; Role; SARS coronavirus; Site; Structural Protein; Temperature; Therapeutic; Time; Vaccines; Viral; Viral Genome; Viral Proteins; Virulent; base; 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.

项目摘要 冠状病毒（COV）已成为强大的人类病原体，因为它们在 人口仅在50年前，是基于快速表征其分子机制的必需品 为了通过治疗剂挫败感染。感染人类的​​七个COV众所周知 致病性，其中包括最近有毒的COV，例如MERS，SARS-COV-1和SARS-COV-2。 COV包括四种结构蛋白，包括包膜（E），膜（M），Spike（S）和 Nucleocapsid（N）蛋白。 COV N蛋白在病毒生命周期中执行许多功能，包括 包装基因组RNA和操纵宿主细胞机械，使N蛋白最多 在感染过程中大量表达病毒蛋白。但是，Cov n的独特和多样化功能 蛋白质制造了标准的结构方法，以解决其分子相互作用的难度。例如， 这种困难包括它们在RNA结合中的滥交，同时与多个结合位点进行参与，以及 固有动态区域的存在被认为对吸引RNA和宿主蛋白是至关重要的。 NMR 提供解决此类挑战的解决方案，因为可以同时表征多种绑定模式 动态和结构上，我们先前已经证明了COV n内的固有动态区域 NMR可以研究蛋白质（SARS-COV-1）。因此，该R21提案的探索性是发展 旨在阐明SARS-COV-2 N蛋白相互作用的分子细节的策略 RNA（AIM 1）和宿主蛋白（AIM 2）。 基于我们的初步研究，我们假设N蛋白内的特定区域具有 首选的RNA结合位点（AIM 1）和宿主细胞环蛋白A结合位点（AIM 2）。我们将解决这些 针对以下目的： 目标1）确定SARS-COV-2 N蛋白如何靶向RNA。生化和生物物理方法 包括NMR在内，将使用NMR识别序列中N蛋白的高亲和力结合位点 源自基因组RNA，并确定在 复合形成。 AIM 2）确定SARS-COV-2 N蛋白如何靶向宿主细胞环蛋白-A。 NMR将习惯 确定N蛋白是否靶向环磷脂A活性位点以及N蛋白是否是底物。