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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结