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

基于机制的 SARS-CoV-2 RNA 加工机制靶向

基本信息

批准号：
10671628
负责人：
Yogesh K Gupta
金额：
$ 58.47万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10671628
关键词：
2019-nCoV ACE2 Ablation Active Sites Address Amino Acids Architecture Artificial Intelligence Bacterial Artificial Chromosomes Binding Biochemical Biological Assay Biology Biophysics COVID-19 COVID-19 therapeutics COVID-19 treatment Cells Chiroptera Communication Complex Coronavirus Crystallization Cultured Cells Dangerousness Data Disease Distant Economics Enzymes Future Genetic Transcription Genome Genomic approach Growth Health Human Immune Immune Evasion Immune response Immune system Individual Infection Innate Immune Response Invaded Investigation K-18 conjugate Length Libraries Life Cycle Stages Maintenance Maps Messenger RNA Methods Methylation Modeling Modification Molecular Molecular Conformation Morbidity - disease rate Mus Mutation Nature Nonstructural Protein Nucleoproteins Nucleotides Organism Paper Pathogenesis Property Proteins Publishing RNA RNA Binding RNA Caps RNA Degradation RNA Folding RNA Processing RNA Viruses RNA chemical synthesis RNA methylation Recombinants Recording of previous events Reporting Research S-Adenosylhomocysteine S-Adenosylmethionine SARS coronavirus SARS-CoV-2 infection SARS-CoV-2 inhibitor Series Solid Specificity Structure Structure-Activity Relationship Surface Technology Testing Time Translations Untranslated Regions Viral Viral Genome Viral Pathogenesis Viral Proteins Virion Virus Virus Assembly Virus Diseases Virus Replication X-Ray Crystallography analog betacoronavirus biophysical analysis combinatorial convolutional neural network drug candidate drug repurposing effective therapy epidemiologic data genetic approach improved in vitro testing in vivo innovation methyl group mortality mouse model novel novel therapeutic intervention operation pandemic disease programs protein complex reverse genetics screening small molecule small molecule libraries stem structural determinants therapeutic target tool transcriptomics vaccine development viral RNA

项目摘要

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).

抽象的大规模的全球大流行，发病率高和死亡率使严重的急性呼吸综合症冠状病毒2（SARS-COV-2）最近历史上最致命的病毒之一。特别值得注意的是劫持人类细胞的正常操作。要开发有效的疗法，我们需要更好的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编码非结构蛋白（NSP）16/NSP10蛋白质复合物，从S-腺苷转运A甲基甲基氨酸（SAM）至病毒mRNA的第一个转录核肠中的2'-OH，从而转换了CAP-0 （M7GPPPA）至CAP-1（M7GPPPAM）。由此产生的病毒mRNA模仿宿主细胞的mRNA。这样，单元不可能区分自己的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，并帮助病毒接管宿主翻译机械以合成其自己蛋白质生存和繁殖。 NSP16活性的消融应引发对病毒的免疫反应感染和限制发病机理。我们最近在自然通讯中的论文描述了原子级别的细节 NSP16/NSP10复合物以及如何很好地适应酶结合RNA盖并发挥2'-OH 甲基化。我们还发现了NSP16中的一个远处的口袋（距催化中心25Å） SARS-COV-2独有。我们还发现，NSP16中的这个口袋部分由 SARS-COV-2独有。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旨在解释对维持RNA基因组和逃避至关重要的因素的术语研究计划从主持人的免疫反应中。我们的研究将揭示SARS-COV-2 RNA CAP的基本原理修改，核蛋白（NP）组装的模式，与mRNA的相互作用以及新方法治疗靶向。在AIM 1中，我们将解决一系列NSP16/NSP10蛋白的新结构通过X射线晶体学转移甲基的每个步骤。结构数据将通过详细信息验证生化和生物物理研究。我们将解决组装的生化和结构决定剂病毒RNA封盖机制，并确定RNA基因组完整性的基本因素。在AIM 2中，我们将开发一种新型的分子工具来研究病毒感染过程中RNA甲基化的临时分布。我们将检查通过药物重新利用或新颖的小型抑制病毒蛋白组合抑制病毒蛋白的新模型分子。最后，我们将根据使用细菌人造染色体（BAC）生成含有NSP16中突变的重组（R）SARS-COV2 确定它们在我们最近使用我们的最近在培养细胞中在培养细胞中的病毒复制和发病机理中的贡献描述了K18人血管紧张素转化酶2（HACE2）SARS-COV-2感染的小鼠模型和相关冠状病毒疾病2019（Covid-19）。