RAPID: Mechanisms of Polymerization Catalyzed by the SARS-CoV-2 RNA Dependent RNA Polymerase

该研究项目旨在填补我们在RNA依赖RNA聚合酶(RdRp)如何准确转录导致新冠肺炎大流行的SARS-CoV-2病毒基因组方面的一个根本空白。该项目的目标是确定基本的RdRp酶和几个合作伙伴蛋白在病毒生命周期中发挥作用的机制。研究成果将使我们更好地了解目前的抗病毒核苷酸类似物，如Remdesivir是如何抑制RdRp功能的，以及未来针对新冠肺炎的新治疗策略的开发。该项目还将支持对两名博士生的培训，这两名博士生都来自STEM领域代表性不足的群体。暂态动力学方法被计划用来研究SARS-CoV-2 RdRp、nsp12和基本辅因子nsp7、nsp8和nsp14催化的核苷酸加成反应。需要检验的一个中心假设是，nsp14核酸外切酶对于提高nsp12聚合酶的保真度是必不可少的。为了解决这一假说，将确定nsp12/nsp7/nsp8复合体催化的正确和错误核苷酸掺入以及nsp14催化的错误核苷酸去除的动力学机制。由此产生的机制知识和方法的发展将更好地将这些蛋白质定义为药物发现的目标。这一快速奖项由分子和细胞生物科学部门的遗传机制计划作出，资金来自冠状病毒援助、救济和经济安全(CARE)法案。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The research project seeks to fill a fundamental gap in our knowledge of how RNA dependent RNA polymerase (RdRp) accurately transcribes the genome of the SARS-CoV-2 virus that has caused the COVID-19 pandemic. The goals of the project are to determine the mechanisms whereby the essential RdRp enzyme and several collaborating partner proteins function in the viral life cycle. The research outcomes will enable better understanding of how current antiviral nucleotide analogs such as Remdesivir inhibit RdRp function, as well as development of new therapeutic strategies against COVID-19 going forward. The project will also support training of two Ph.D. students, both of whom are from underrepresented groups in STEM fields. Transient state kinetic approaches are planned to interrogate the nucleotide addition reaction catalyzed by SARS-CoV-2 RdRp, nsp12, and the essential cofactors nsp7, nsp8, and nsp14. A central hypothesis to be tested is that the nsp14 exonuclease is essential for increased fidelity of the nsp12 polymerase. To address this hypothesis, the kinetic mechanisms of correct and incorrect nucleotide incorporation catalyzed by nsp12/nsp7/nsp8 complex as well as removal of incorrect nucleotides catalyzed by nsp14 will be determined. The resulting mechanistic knowledge and methods development will better define these proteins as targets for drug discovery.This RAPID award is made by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.