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：
10629420
负责人：
SERGEI BORUKHOV
金额：
$ 22.78万
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 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10629420
关键词：
2019-nCoV Address Antiviral Agents Binding Biochemical Biological Assay COVID-19 COVID-19 pandemic Catalytic Domain Cells Complex Elements Engineering Event Exhibits Exonuclease Fluorescence Gel Genetic Transcription Goals Human Hydroxyl Radical In Vitro Incubated Label Laboratories Length Maps Mediating Messenger RNA Methods Molecular 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 Nonstructural Proteins Viral Proteins antiviral drug development design genomic RNA guanylate helicase in vivo pathogen protein crosslink recombinant RNA reconstitution single molecule single-molecule FRET targeted agent uridylate viral RNA

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/摘要该合作项目的总体目标是了解启动和发生的分子机制。冠状病毒 SARS-CoV-2 RNA 依赖性 RNA 聚合酶 (RdRp) 的不连续 RNA 合成为此，Sergei Borukhov 博士的实验室建立了调节这些过程的决定因素。罗文大学 SOM 和加州大学洛杉矶分校的 Shimon Weiss 博士将：(i) 重建体外最小的 SARS-CoV2 转录/复制系统能够独立于引物（从头）和蛋白质引发（-）和 (+) 使用病毒 RdRp、5'-UTR 和 3'-UTR RNA 元件合成 RNA 链，并进行核苷酸化（尿苷化） (ii) 描述病毒传播过程生物化学转录/复制起始并确定病毒蛋白因子（nsp9、 nsp10、nsp13、nsp14 和 N 蛋白），使用基于凝胶的整体检测以及最近开发的单 (iii) 重建 SARS-CoV-2 不连续转录系统，其中 RdRp 在 RNA 合成过程中在转录调节序列 (TRS) 处暂停并切换 RNA 产生嵌套的亚基因组 mRNA 的模板；(iv) 开发单分子和整体水平测定，例如基于单分子 FRET 的模板转换测定、RNA-蛋白质交联、核酸外切酶足迹和局部 Fe2+ 诱导的羟基自由基作图来表征不连续转录中间体（例如暂停、暂停回溯和模板切换 RdRp (v) 鉴定模板转换所需的病毒和人类宿主细胞蛋白因子。