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

项目总结 冠状病毒(CoV)自被发现以来，已成为强大的人类病原体 人类种群仅在50年前，潜在的需要快速表征他们的分子机制 以便通过治疗来阻止他们的感染。已知七种感染人类的冠状病毒具有 致病性，其中包括最近的强毒冠状病毒，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)、穗(S)和 核衣壳蛋白。冠状病毒N蛋白在病毒生命周期中发挥多种功能，包括 包装基因组RNA和操纵宿主细胞机制，使N蛋白成为最 在感染过程中大量表达病毒蛋白。然而，冠状病毒N的独特和多样化的功能 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结合中的混杂，同时接触多个结合位点，以及 固有的动态区域的存在被认为对RNA和宿主蛋白质的结合至关重要。核磁共振 为此类挑战提供了解决方案，因为多种绑定模式可以同时表征这两种模式 从动力学和结构上讲，我们之前已经证明了覆盖层N内的固有动态区域 蛋白质(SARS-CoV-1)可以用核磁共振进行研究。因此，这项R21提案的探索性性质是开发 旨在阐明SARS-CoV-2N蛋白与其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 首选RNA结合位点(AIM 1)和宿主细胞亲环素-A结合位点(AIM 2)。我们将解决这些问题 通过以下方式实现目标： 目的1)确定SARS-CoV-2N蛋白是如何靶向RNA的。生化和生物物理方法 包括核磁共振，将用于鉴定序列中N蛋白的高亲和力结合位点 从基因组RNA中获得，并识别发生在 复杂的队形。 目的2)确定SARS-CoV-2N蛋白如何靶向宿主细胞亲环素-A。核磁共振将被用于 确定N蛋白是否以亲环素-A活性部位为靶标，以及N蛋白是否为底物。

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.