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的全球大流行具有高发病率和死亡率 冠状病毒-2 (SARS-CoV-2) 是近代历史上最致命的病毒之一。尤其值得注意的是 劫持人体细胞的正常运作。为了开发有效的治疗方法，我们需要更好地了解 允许病毒侵入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 (nsp)16/nsp10 蛋白复合物 甲硫氨酸 (SAM) 转化为病毒 mRNA 第一个转录核苷酸的 2’-OH，从而将 Cap-0 转化为 (m7GpppA) 到 Cap-1 (m7GpppAm)。由此产生的病毒 mRNA 模仿宿主细胞的 mRNA。这样，细胞就不能 区分自身的RNA和病毒的RNA。病毒编码的 mRNA 的这种修饰不仅 欺骗免疫系统并帮助病毒接管宿主翻译机器以合成自己的翻译机器 用于生存和繁殖的蛋白质。 nsp16 活性的消除应该会引发对病毒的免疫反应 感染并限制发病机制。我们最近在《自然通讯》上发表的论文描述了原子级细节 nsp16/nsp10 复合物以及酶如何很好地适应结合 RNA 帽并发挥 2'-OH 甲基化。我们还在 nsp16 中发现了一个遥远的口袋（距离催化中心 25Å），即 SARS-CoV-2 所特有的。我们还发现 nsp16 中的这个口袋部分由氨基酸组成 SARS-CoV-2 所特有的。它可以结合催化中心外的小分子。我们建议建立一个长期 旨在破译对RNA基因组维持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) 组装模式、与 mRNA 的相互作用以及新的方法 治疗靶向。在目标 1 中，我们将解析捕获的 nsp16/nsp10 蛋白的一系列新结构。 通过 X 射线晶体学分析甲基转移的每一步。结构数据将通过详细的验证 生物化学和生物物理研究。我们将解决装配的生化和结构决定因素 病毒RNA加帽机制，并确定RNA基因组完整性的潜在因素。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使用的反向遗传学方法 细菌人工染色体 (BAC) 生成重组 (r)SARS-CoV2，其中包含 nsp16 突变 使用我们最近的研究确定它们在培养细胞中的病毒复制和体内发病机制中的贡献 描述了 SARS-CoV-2 感染的 K18 人血管紧张素转换酶 2 (hACE2) 小鼠模型和 相关的 2019 年冠状病毒病 (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).