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: 10238209
• 负责人: ELIZABETH A CAMPBELL
• 金额: $ 63.81万
• 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/10238209
• 关键词: 2019-nCoV; Active Sites; Antiviral Agents; Architecture; Biochemical; Biological Assay; COVID-19; COVID-19 pandemic; Catalysis; Cells; Cessation of life; Classification; Collaborations; Color; Complex; Coronavirus; 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; Genomics; Goals; Hand; Higher Order Chromatin Structure; Holoenzymes; Human Microbiome; In Vitro; Infection; Investigation; Lead; Manuscripts; Maps; Mass Spectrum Analysis; Methyltransferase; Modeling; Molecular Conformation; Molecular Structure; N-terminal; Nucleic Acids; Nucleotides; RNA; RNA Polymerase I; RNA Polymerase Inhibitor; RNA Viruses; RNA replication; 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; Virus; Virus Replication; Water; antimicrobial; base; drug discovery; endonuclease; experimental study; helicase; illness length; improved; in vivo; inhibitor/antagonist; insight; large datasets; new therapeutic target; novel; novel therapeutics; nucleotide analog; pathogen; protein protein interaction; remdesivir; single molecule; single-molecule FRET; stoichiometry; viral RNA

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.

项目摘要 Covid-19，由冠状病毒SARS-COV-2引起，继续破坏世界。在不到一年的时间内 有超过70万人死亡的案件超过2000万例。病毒RNA依赖性RNA 聚合酶（RDRP）是负责转录和复制病毒RNA的中心酶 基因组。该酶也是当前抗病毒Remdesivir的靶标，用于改善严重程度和 这种疾病的持续时间。该病毒还编码了几种核酸加工酶 RDRP，包括解旋酶，核酸内切酶，外切核酸酶和甲基转移酶。但是，是 未知这些酶如何协调转录和复制病毒基因组。该建议建立 根据解旋酶结构NSP13的初步数据，与RDRP和启动底物复杂 RNA（NSP13-复制/转录复合物或NSP13-RTC）。这里的目的包括完成结构 通过使用收集的其他数据来分析该复合物。这个目标的结果将提供更高的 分辨率（在RDRP的某些地方比2.7Å更好），为抗病毒的发展提供了丰富的基础 抑制剂。此外，拥有这种结构可以与 抗菌剂也是目标的一部分，包括投资前结构细节的投资 由人类微生物组产生的remdesivir和抗病毒药的状态。 由NSP13-RTC的结构产生的模型是测试解旋酶和方式的基础 外核酸酶与RDRP一起功能。具体而言，实时荧光测定，单分子 荧光共振能量传递（FRET）和多色荧光显微镜将用于证明 解旋酶和外切酶在放松底物RNA，回溯和校对中的作用。 另一个目标应用用于表征NSP13-RTC组件的管道，该组件产生了高度 复合物的分辨率结构，到其他RTC组件。具体而言，天然电泳迁移率 测定将用作探测RTC较大组件的起点。天然质谱法会 然后用于确定复合物的组成和化学计量。最后，Cryo-Em将是 应用于解决这些大分子机器的结构。由此产生的结构将提供 阐明这些酶的协调功能的起点，提供了对其机制的见解， 并建立新的治疗目标。 总而言之，该提议旨在在分子和结构层面了解SARS-COV-2如何了解 核酸加工酶坐标以复制和转录病毒基因组，并提供 结构引导的药物发现目标，其最终目的是为Covid-19提供缓解的目标 大流行。