Untangling the mechanisms of initiation and discontinuous RNA synthesis by COVID-19 RdRp

解开 COVID-19 RdRp 启动和不连续 RNA 合成的机制

基本信息

批准号：
10511943
负责人：
SERGEI BORUKHOV
金额：
$ 20.29万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10511943
关键词：
2019-nCoV Address Binding Biochemical Biological Assay COVID-19 COVID-19 pandemic Catalytic Domain Cells Complex Coronavirus Elements Engineering Event Exhibits Exonuclease Fluorescence Gel Genetic Transcription Genomics Goals Human Hydroxyl Radical In Vitro Incubated Label Laboratories Length Mediating Messenger RNA Methods Molecular Nonstructural Protein Nucleotides Open Reading Frames Positioning Attribute Process Proteins RNA RNA chemical synthesis RNA primers RNA replication RNA-Directed RNA Polymerase Reaction Replication Initiation Reporter Role Sorbus Structure System Testing Transcription Initiation Universities Untranslated Regions Viral Viral Proteins antiviral drug development base crosslink design genomic RNA guanylate helicase in vivo oligomycin sensitivity-conferring protein pathogen recombinant RNA reconstitution single molecule single-molecule FRET targeted agent uridylate viral RNA

2019-nCoV Address Binding Biochemical Biological Assay COVID-19 COVID-19 pandemic Catalytic Domain Cells Complex Coronavirus Elements Engineering Event Exhibits Exonuclease Fluorescence Gel Genetic Transcription Genomics Goals Human Hydroxyl Radical In Vitro Incubated Label Laboratories Length Mediating Messenger RNA Methods Molecular Nonstructural Protein Nucleotides Open Reading Frames Positioning Attribute Process Proteins RNA RNA chemical synthesis RNA primers RNA replication RNA-Directed RNA Polymerase Reaction Replication Initiation Reporter Role Sorbus Structure System Testing Transcription Initiation Universities Untranslated Regions Viral Viral Proteins antiviral drug development base crosslink design genomic RNA guanylate helicase in vivo oligomycin sensitivity-conferring protein pathogen recombinant RNA reconstitution single molecule single-molecule FRET targeted agent uridylate viral RNA

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项目摘要

SUMMARY/ ABSTRACT The broad goal of this collaborative project is to understand the molecular mechanisms of initiation and discontinuous RNA synthesis by the coronavirus SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and establish determinants that regulate these processes. To this end, the laboratories of Dr. Sergei Borukhov at Rowan University SOM and of Dr. Shimon Weiss at UCLA will: (i) reconstitute an in vitro minimal SARS-CoV2 transcription/replication system capable of primer-independent (de novo) and protein-primed initiation of (-) and (+) RNA strand synthesis using viral RdRp, 5’-UTR and 3’-UTR RNA elements, and nucleotidylated (uridylated and guanylated) viral non-structural proteins nsp8 and nsp9; (ii) characterize the process of viral transcription/replication initiation biochemically and determine the contribution of viral protein factors (nsp9, nsp10, nsp13, nsp14, and N protein), using gel-based ensemble assays as well as recently developed single- molecule RdRp activity assays; (iii) reconstitute SARS-CoV-2 discontinuous transcription system in which RdRp pauses during RNA synthesis at transcription-regulating sequences (TRSs) and switches RNA templates to produce nested sets of sub-genomic mRNAs; (iv) develop both single-molecule and ensemble level assays such as single-molecule FRET based template switching assay, RNA-protein cross-linking, exonuclease-footprinting and localized Fe2+ -induced hydroxyl-radical mapping to characterize the discontinuous transcription intermediates (e.g., paused, paused-backtracked, and template-switched RdRp complexes); (v) identify viral and human host cell protein factors that are required for template-switching.

摘要/摘要这个协作项目的广泛目标是了解主动性的分子机制和冠状病毒SARS-COV-2 RNA依赖性RNA聚合酶（RDRP）和确定调节这些过程的决定。为此，Sergei Borukhov博士的实验室在 UCLA的Rowan University SOM和Shimon Weiss博士将：（i）重建一个体外的SARS-COV2 能够独立于底漆的转录/复制系统（从头开始）和蛋白质提出的倡议（ - ）和（+）使用病毒RDRP，5'-UTR和3'-UTR RNA元素以及核苷酸化（尿苷）的RNA链合成（尿酸）和鸟苷）病毒非结构性蛋白NSP8和NSP9；（ii）表征病毒的过程转录/复制倡议在生物化学上，并确定病毒蛋白因子的贡献（NSP9，NSP9， NSP10，NSP13，NSP14和N蛋白），使用基于凝胶的集合测定以及最近开发的单个单个分子RDRP活性评估；（iii）重构SARS-COV-2不连续的转录系统，其中在转录调节序列（TRS）的RNA合成期间的RDRP暂停并开关RNA 产生亚基因组mRNA嵌套集的模板；（iv）同时发展单分子和合奏水平测定，例如基于单分子FRET的模板开关测定，RNA-蛋白交联，外切核酸酶打印和局部Fe2+诱导的羟基 - 自由基映射以表征不连续的转录中间体（例如，暂停，暂停折磨和模板切换的RDRP 复合体）; （v）确定模板切换所需的病毒和人类宿主细胞蛋白因子。