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对 RNA 依赖性 RNA 聚合酶 (RdRp) 如何准确转录导致 COVID-19 大流行的 SARS-CoV-2 病毒基因组的认识的根本空白。该项目的目标是确定必需的 RdRp 酶和几种协作伙伴蛋白在病毒生命周期中发挥作用的机制。研究成果将有助于更好地了解当前的抗病毒核苷酸类似物（例如瑞德西韦）如何抑制 RdRp 功能，以及开发针对未来 COVID-19 的新治疗策略。该项目还将支持培养两名博士生。学生，他们都来自 STEM 领域代表性不足的群体。计划采用瞬态动力学方法来研究 SARS-CoV-2 RdRp、nsp12 以及必需辅因子 nsp7、nsp8 和 nsp14 催化的核苷酸加成反应。要测试的一个中心假设是 nsp14 核酸外切酶对于提高 nsp12 聚合酶的保真度至关重要。为了解决这一假设，将确定由 nsp12/nsp7/nsp8 复合物催化的正确和不正确的核苷酸掺入以及由 nsp14 催化的不正确的核苷酸去除的动力学机制。由此产生的机械知识和方法开发将更好地将这些蛋白质定义为药物发现的靶标。该 RAPID 奖项由分子和细胞生物科学部的遗传机制计划使用冠状病毒援助、救济和经济安全 (CARES) 法案的资金颁发。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值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.