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

项目摘要 冠状病毒（CoV）自从在体内被检测到以来，已经成为可怕的人类病原体。 人类人口只有50年前，根本需要迅速表征其分子机制 以通过治疗来阻止他们的感染。已知有七种感染人类的冠状病毒 致病性，其中包括最近的毒性冠状病毒，如MERS，SARS-CoV-1和SARS-CoV-2。 CoV包含四种结构蛋白，包括包膜（E）、膜（M）、刺突（S）和膜（M）。 核衣壳（N）蛋白。CoV N蛋白在病毒的生命周期中执行许多功能，包括 包装基因组RNA和操纵宿主细胞机器，使N蛋白成为最重要的 在感染过程中大量表达病毒蛋白。然而，CoV N独特而多样的功能 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。比如说， 这些困难包括它们在RNA结合中的混杂性，同时接合多个结合位点， 存在被认为对接合RNA和宿主蛋白质至关重要的固有动态区域。NMR 提供了一个解决方案，因为多种绑定模式可以同时表征， 动态和结构，我们以前已经表明，在一个冠状病毒N内的固有动态区域， 蛋白质（SARS-CoV-1）可以通过NMR进行研究。因此，这项R21提案的探索性质是， 这些策略旨在阐明SARS-CoV-2 N蛋白与 RNA（Aim 1）和宿主蛋白（Aim 2）。 基于我们的初步研究，我们假设N蛋白中的特定区域具有 优选的RNA结合位点（Aim 1）和宿主细胞亲环蛋白-A结合位点（Aim 2）。我们将解决这些问题 目标如下： 目的1）确定SARS冠状病毒2型N蛋白如何靶向RNA。生物化学和生物物理方法 包括NMR在内的技术将用于鉴定序列内N蛋白的高亲和力结合位点 来自基因组RNA，并确定相关的动态和结构变化， 复杂的形成。 目的2）确定SARS冠状病毒2型N蛋白如何靶向宿主细胞亲环素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蛋白是否是底物。

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.