Mechanism-based Targeting of the RNA Processing Machinery of SARS-CoV-2

摘要 高发病率和高死亡率的大规模全球大流行使严重急性呼吸综合征 冠状病毒-2(SARS-CoV-2)是近年来最致命的病毒之一。尤其值得注意的是 劫持人类细胞的正常运作。为了开发有效的治疗方法，我们需要更好地理解 允许病毒入侵细胞并逃避宿主免疫限制的机制。SARS-CoV-2编码 从S-腺苷转移甲基的非结构蛋白16/NSP10蛋白复合体 将甲硫氨酸(SAM)转化为病毒mRNA的第一转录核苷酸的2‘-OH，从而将Cap-0 (M7GpppA)到Cap-1(M7GpppAm)。由此产生的病毒信使核糖核酸模仿宿主细胞的信使核糖核酸。这样，单元格就不能 区分病毒自身的RNA和病毒的RNA。病毒编码的信使核糖核酸的这种修饰不仅 欺骗免疫系统，帮助病毒接管宿主的翻译机器，进行自身的合成 生存和繁殖所需的蛋白质。Nsp16活性的消融应该会触发对病毒的免疫反应 感染和限制发病机制。我们最近在《自然通讯》上的论文描述了原子级的细节 Nsp16/nsp10复合体以及酶如何很好地结合RNA帽并作用于2‘-OH 甲基化。我们还在nsp16中发现了一个远离催化中心的口袋(距离催化中心25？)，即 这是SARS-CoV-2独有的。我们还发现，nsp16中的这个口袋部分由以下氨基酸组成 这是SARS-CoV-2独有的。它可以结合催化中心外的小分子。我们建议建造一座长长的- 旨在破译对维持RNA基因组和逃避至关重要的因素的学期研究计划 来自宿主的免疫反应。我们的研究将揭示SARS-CoV-2 RNA帽的基本原理 修饰，核蛋白(NP)的组装方式，与mRNA的相互作用，以及新的途径 治疗靶向。在目标1中，我们将解析nsp16/nsp10蛋白的一系列新结构，这些蛋白在 用X射线结晶学分析了甲基转移的每一步。结构数据将通过详细的 生化和生物物理研究。我们将解决组装的生化和结构决定因素 病毒RNA封顶机制，并确定潜在的RNA基因组完整性的因素。在目标2中，我们将 开发一种新的分子工具来研究病毒感染过程中RNA甲基化的时间分布。我们 将研究通过药物再利用或新的小分子药物组合抑制病毒蛋白的新模型 分子。最后，我们将使用我们最近建立的基于 细菌人工染色体(BAC)产生含有nsp16突变的重组SARS-CoV2 利用我们的最新研究结果确定它们在培养细胞病毒复制和体内致病机制中的作用 描述了K18人血管紧张素转换酶2(HACE2)感染SARS-CoV-2小鼠模型和 2019年相关冠状病毒病(新冠肺炎)。

ABSTRACT The massive global pandemic with high morbidity and mortality makes Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) one of the deadliest viruses in recent history. It is especially noteworthy for hijacking the normal operations of human cells. To develop effective therapies, we need a better understanding of the mechanisms that permit the virus to invade cells and evade host immune restriction. SARS-CoV-2 encodes the non-structural protein (nsp)16/nsp10 protein complex that transfers a methyl group from S-adenosyl methionine (SAM) to 2’-OH of the first transcribing nucleotide of the viral mRNA and thus converts the Cap-0 (m7GpppA) to Cap-1 (m7GpppAm). The resulting viral mRNA mimics host cell’s mRNA. In this way, a cell cannot distinguish between its own RNA and that of the virus. This modification of the virally encoded mRNA not only tricks the immune system and helps the virus to take over the host translation machinery for synthesis of its own proteins for survival and propagation. Ablation of nsp16 activity should trigger an immune response to viral infection and limit pathogenesis. Our recent paper in Nature Communications described atomic level details of the nsp16/nsp10 complex and how the enzyme is well adapted to bind the RNA cap and exert the 2’-OH methylation. We also discovered a distant pocket (located 25Å away from the catalytic center) in nsp16 that is unique to SARS-CoV-2. We also found that this pocket in nsp16 is partially composed of amino acids that are unique to SARS-CoV-2. It can bind small molecules outside of the catalytic center. We propose to build a long- term research program aimed at deciphering the factors crucial to the maintenance of RNA genome and evasion from the host’s immune response. Our studies will reveal basic principles underlying SARS-CoV-2 RNA cap modification, the mode of nucleoprotein (NP) assembly, interplay with mRNA, and new approaches for therapeutic targeting. In Aim 1, we will resolve a series of new structures of nsp16/nsp10 proteins captured in every step of the methyl transfer by X-ray crystallography. The structural data will be validated by detailed biochemical and biophysical studies. We will resolve the biochemical and structural determinants of the assembly of viral RNA capping machinery, and identify factors underlying integrity of RNA genome. In Aim 2, we will develop a novel molecular tool to study temporal distribution of the RNA methylation during viral infection. We will examine new models for combinatorial inhibition of viral proteins by drug repurposing or novel small molecules. Finally, we will use our recently established reverse genetics approaches based on the use of a bacterial artificial chromosome (BAC) to generate recombinant (r)SARS-CoV2 containing mutations in nsp16 to determine their contribution in viral replication in cultured cells and pathogenesis in vivo using our recently described K18 human angiotensin converting enzyme 2 (hACE2) mouse model of SARS-CoV-2 infection and associated coronavirus disease 2019 (COVID-19).