Structure, function, and inhibition of the SARS-CoV-2 replication-transcription complex

SARS-CoV-2 复制转录复合物的结构、功能和抑制

• 批准号: 10669076
• 负责人: ELIZABETH A CAMPBELL
• 金额: $ 62.05万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-06 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10669076
• 关键词: 2019-nCoV; Active Sites; Antiviral Agents; Architecture; Binding; Biochemical; Biological Assay; COVID-19; COVID-19 pandemic; Catalysis; Cells; Cessation of life; Classification; Collaborations; Color; Complex; Cryoelectron Microscopy; Data; Data Set; Development; Diphosphates; Disease; Drug Design; Drug Targeting; Effectiveness; Electrophoretic Mobility Shift Assay; Enzymes; Exonuclease; Fluorescence; Fluorescence Microscopy; Foundations; Gel; Gene Expression; Genetic Transcription; Genome; Goals; Hand; Higher Order Chromatin Structure; Holoenzymes; Human Microbiome; In Vitro; Infection; Investigation; Manuscripts; Maps; Mass Spectrum Analysis; Methyltransferase; Modeling; Molecular; Molecular Conformation; Molecular Structure; N-terminal; Nucleic Acids; Nucleotide Mapping; Polymerase; RNA Polymerase Inhibitor; RNA-Directed RNA Polymerase; Reaction; Replication-Associated Process; Reporting; Resolution; Rice; Role; SARS-CoV-2 genome; SARS-CoV-2 inhibitor; Scientist; Severities; Site; Structure; Testing; Time; Transcription Process; Viral; Viral Genome; Viral Physiology; Virus; Virus Inhibitors; Virus Replication; Water; antimicrobial; antiviral drug development; cofactor; drug discovery; endonuclease; experimental study; helicase; illness length; improved; in vivo; inhibitor; insight; large datasets; new therapeutic target; novel; novel therapeutics; nucleotide analog; pathogen; protein protein interaction; remdesivir; single molecule; single-molecule FRET; stoichiometry

Project Summary COVID-19, caused by the coronavirus SARS-CoV-2, continues to devastate the world. In less than a year, there have been more than 20 million cases with over 700,000 deaths. The viral RNA-dependent RNA polymerase (RdRp) is the central enzyme responsible for transcription and replication of the viral RNA genome. This enzyme is also a target for the current antiviral, remdesivir, used to ameliorate the severity and duration of this disease. The virus also encodes several nucleic acid processing enzymes, in addition to the RdRp, including a helicase, an endonuclease, an exonuclease, and methyltransferases. However, it is unknown how these enzymes coordinate to transcribe and replicate the viral genome. This proposal builds upon preliminary data of the structure of the helicase, nsp13, in complex with the RdRp and a primed substrate RNA (nsp13-replication/transcription complex or nsp13-RTC). The aims here include completing the structural analysis of this complex by utilizing additional data collected. The result of this aim will provide higher resolution (better than 2.7 Å in some parts the RdRp), providing a rich basis for the development of antiviral inhibitors. Also, having this structure in hand allows for the collaboration with expert developers of antimicrobials, also part of the aims, including the investigation of the structural details of the pre-incorporation state of remdesivir and antivirals produced by human microbiome. The models resulting from the structure of nsp13-RTC serve as foundations to test how the helicase and exonuclease function together with the RdRp. Specifically, real-time fluorescence assays, single-molecule fluorescence resonance energy transfer (FRET), and multi-color fluorescence microscopy will be used to probe the role of the helicase and the exonuclease in unwinding substrate RNA, backtracking, and proofreading. Another aim applies the pipeline used to characterize the nsp13-RTC assembly, which yielded a high- resolution structure of the complex, to other RTC assemblies. Specifically, native electrophoretic mobility assays will be used as a starting point to probe larger assemblies of the RTC. Native mass-spectrometry will then be used to determine the composition and stoichiometry of the complexes. Finally, cryo-EM will be applied to solve the structures of these macromolecular machines. The resulting structures will provide a starting point to elucidate the coordinated functions of these enzymes, provide insight into their mechanisms, and establish novel targets for therapeutics. In summary, this proposal aims to understand at the molecular and structural level how the SARS-CoV-2 nucleic acid processing enzymes coordinate to replicate and transcribe the viral genome, and to provide structure-guided targets for drug discovery, with the ultimate goal of providing relief for the COVID-19 pandemic.

项目概要 由冠状病毒 SARS-CoV-2 引起的 COVID-19 继续摧毁世界。在不到一年的时间里， 确诊病例已超过2000万例，死亡人数超过70万人。病毒RNA依赖性RNA 聚合酶 (RdRp) 是负责病毒 RNA 转录和复制的中心酶 基因组。这种酶也是当前抗病毒药物瑞德西韦（remdesivir）的靶标，用于缓解严重程度和 这种疾病的持续时间。除了编码酶外，该病毒还编码多种核酸加工酶。 RdRp，包括解旋酶、核酸内切酶、核酸外切酶和甲基转移酶。然而，它是 未知这些酶如何协调转录和复制病毒基因组。该提案构建 根据解旋酶 nsp13 与 RdRp 和底物的复合物结构的初步数据 RNA（nsp13-复制/转录复合物或 nsp13-RTC）。这里的目标包括完成结构 利用收集到的额外数据对该复合物进行分析。这一目标的结果将提供更高 分辨率（RdRp的某些部分优于2.7 Å），为抗病毒药物的开发提供了丰富的基础 抑制剂。此外，掌握此结构还可以与以下领域的专家开发人员进行协作： 抗菌药物，也是目标的一部分，包括研究预掺入的结构细节 瑞德西韦和人类微生物组产生的抗病毒药物的状态。 由 nsp13-RTC 结构产生的模型可作为测试解旋酶和 核酸外切酶与 RdRp 一起发挥作用。具体来说，实时荧光测定、单分子 荧光共振能量转移（FRET）和多色荧光显微镜将用于探测 解旋酶和核酸外切酶在解旋底物 RNA、回溯和校对中的作用。 另一个目标是应用用于表征 nsp13-RTC 组件的管道，该组件产生了高 复杂的分辨率结构，以供其他 RTC 组件使用。具体来说，本机电泳迁移率 检测将用作探测 RTC 更大组件的起点。天然质谱将 然后用于确定复合物的组成和化学计量。最后，冷冻电镜将 应用于解决这些高分子机器的结构。由此产生的结构将提供 阐明这些酶的协调功能的起点，深入了解它们的机制， 并建立新的治疗靶点。 总之，该提案旨在从分子和结构层面了解 SARS-CoV-2 如何 核酸加工酶协调复制和转录病毒基因组，并提供 以结构为导向的药物发现目标，最终目标是缓解 COVID-19 大流行。