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

项目摘要 由冠状病毒SARS-CoV-2引起的COVID-19继续困扰世界。在不到一年的时间里 已经有超过两千万个病例，超过七十万人死亡。的病毒rna依赖性RNA 聚合酶（RdRp）是负责病毒RNA转录和复制的中心酶 基因组这种酶也是目前抗病毒药物remdesivir的靶点，用于改善严重程度， 这种疾病的持续时间。该病毒还编码几种核酸加工酶，除了 RdRp，包括解旋酶、内切核酸酶、外切核酸酶和甲基转移酶。但据 这些酶如何协调转录和复制病毒基因组尚不清楚。这一建议建立 根据解旋酶nsp 13与RdRp和引发底物复合的结构的初步数据， RNA（nsp 13-复制/转录复合物或nsp 13-RTC）。目标包括完成结构 通过使用收集的额外数据对该综合体进行分析。这一目标的结果将提供更高的 分辨率（在某些部分优于2.7 μ m的RdRp），为开发抗病毒药物提供了丰富的基础 抑制剂的此外，拥有这种结构允许与专家开发人员合作， 抗菌剂，也是目标的一部分，包括调查预掺入的结构细节， Remdesivir和人类微生物组产生的抗病毒药物的状态。 由nsp 13-RTC结构产生的模型作为基础来测试解旋酶和 外切核酸酶与RdRp一起起作用。具体地，实时荧光测定、单分子荧光测定、单分子荧光测定。 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。 另一个目的是应用用于表征nsp 13-RTC组装的流水线，其产生了高- 复杂的分辨率结构，其他RTC组件。具体而言，天然电泳迁移率 分析将用作探测RTC的较大组件的起点。原生质谱将 然后用于确定络合物的组成和化学计量。最后，冷冻EM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 起点阐明这些酶的协调功能，提供深入了解其机制， 并建立新的治疗靶点。 总之，这项提议旨在从分子和结构水平上了解SARS-CoV-2如何与病毒结合， 核酸加工酶协调复制和转录病毒基因组，并提供 用于药物发现的结构导向靶标，最终目标是为COVID-19提供缓解 流行病

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.