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

摘要 严重急性呼吸系统综合症（SARS）在全球大规模流行，发病率和死亡率很高， 冠状病毒-2（SARS-CoV-2）是近年来最致命的病毒之一。特别值得注意的是， 劫持人类细胞的正常运作。为了开发有效的治疗方法，我们需要更好地了解 病毒侵入细胞和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（nsp）16/nsp 10蛋白复合物 在一个实施方案中，所述方法包括将病毒mRNA的第一转录核苷酸的甲硫氨酸（SAM）转化为2 '-OH，从而将Cap-0 （m7 GpppA）至Cap-1（m7 GpppAm）。所得的病毒mRNA模拟宿主细胞的mRNA。这样，细胞就不能 区分自己的RNA和病毒的RNA病毒编码的mRNA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， 生存和繁殖的蛋白质。nsp 16活性的消除应该触发对病毒的免疫应答。 感染和有限发病机制。我们最近在《自然通讯》上发表的论文描述了 nsp 16/nsp 10复合物以及酶如何很好地适应于结合RNA帽并施加2 '-OH 甲基化我们还在nsp 16中发现了一个遥远的口袋（位于距离催化中心25英里处）， 是SARS冠状病毒2型独有的我们还发现，nsp 16中的这个口袋部分由氨基酸组成， 是SARS冠状病毒2型独有的它可以结合催化中心外的小分子。我们建议建立一个长期的- 一项长期研究计划，旨在破译对RNA基因组的维持和逃避至关重要的因素 免疫系统的反应。我们的研究将揭示SARS-CoV-2 RNA帽的基本原理 修饰，核蛋白（NP）组装的模式，与mRNA的相互作用，以及新的方法， 治疗靶向在目标1中，我们将解析一系列新结构的nsp 16/nsp 10蛋白， 甲基转移的每一步。结构数据将通过详细的 生物化学和生物物理学研究。我们将解决组装的生化和结构决定因素 病毒RNA加帽机制，并确定RNA基因组完整性的基础因素。在目标2中，我们 开发一种新的分子工具来研究病毒感染期间RNA甲基化的时间分布。我们 将研究通过药物再利用或新型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反向遗传学方法， 细菌人工染色体（BAC），以产生含有nsp 16突变的重组（r）SARS-CoV 2， 确定他们的贡献，病毒复制在培养细胞和发病机制，在体内使用我们最近的 描述了SARS-CoV-2感染的K18人血管紧张素转换酶2（hACE 2）小鼠模型， 2019冠状病毒病（COVID-19）。

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).