RAPID: IIBR: Instrumentation: Time-resolved studies of the SARS-CoV-2 endonuclease NP15

2019年冠状病毒病（COVID-19）危机使人们迫切需要了解导致这种严重传染病的冠状病毒SARS-CoV-2 （CoV-2）的蛋白质结构和动力学。该奖项支持的研究将揭示最有趣和独特的CoV-2酶之一RNA核糖核酸内切酶（NendoU）的催化机制，这种酶只存在于高毒力的冠状病毒中。NendoU在限制宿主传感器对病毒RNA的感知方面起着关键作用，因此是在分子水平上阐明病毒发病机制的一个非常重要的靶点。研究人员将结合先进的技术和样品制备的新创新来制作“分子电影”，揭示NendoU蛋白的作用机制，以促进我们对病毒生物学的理解，并可能有助于识别潜在的治疗化合物。这些研究的结果将通过在线研讨会、科学会议、公共数据库和同行评议期刊的出版物进行分享。在低温条件下，通过标准晶体学确定了来自SARS、MERS以及最近来自CoV-2的NendoU的静态结构，但没有捕获底物RNA结合的结构，也没有对酶进行任何时间分辨率的研究。COVID-2019 RAPID奖侧重于使用时间分辨飞秒晶体学（TR-SFX）和时间分辨低温电子显微镜（TR-cryo-EM）相结合的时间分辨研究，以捕捉NendoU催化反应的分子电影。在建立NendoU的微/纳米结晶条件后，将在x射线自由电子激光器（XFEL）上进行混合注入TR-SFX研究，以捕获关键反应步骤的快照，包括底物结合和催化反应。其次，为Tr-SFX开发的混合注射技术将适用于TR-cryo-EM，该技术通过揭示催化后期时间点（包括产物释放和稳态）来补充Tr-SFX研究。这些具有时间分辨率的NendoU研究将揭示过渡态的结构，并可能为开发新型冠状病毒严重感染药物或研究NendoU在抑制免疫反应中的作用提供基础。亚利桑那州立大学的这项RAPID奖是由生物基础设施部利用《冠状病毒援助、救济和经济安全法案》的资金颁发的。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识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 coronavirus disease 2019 (COVID-19) crisis has produced an urgent need to understand the structure and dynamics of the proteins of SARS-CoV-2 (CoV-2), the coronavirus that is the cause of this severe infectious disease. Research supported by this award will unravel the catalytic mechanisms of one of the most interesting and unique CoV-2 enzymes, the RNA endoribonuclease, NendoU, which is only found in the highly virulent coronaviruses. NendoU plays a key role in limiting the sensing of viral RNA by host sensors and is thus a very important target to elucidate the mechanisms of viral pathogenesis at the molecular level. The researchers will use a combination of advanced techniques and new innovations in sample preparation to make “molecular movies” that reveal the mechanism of action for the NendoU protein to advance our understanding of viral biology and may aid in the identification of potential therapeutic compounds. Results from these studies will be shared through on-line seminars, at scientific meetings, in public data repositories, and through publications in peer-reviewed journals.Static structures of the NendoU from SARS, MERS and most recently, also from CoV-2 have been determined by standard crystallography under cryogenic conditions, but no structures have been captured with the substrate RNA bound nor have any time-resolved studies been performed on the enzyme. This COVID-2019 RAPID award focuses on time resolved studies using a combination of time-resolved femtosecond crystallography (TR-SFX) and time resolved cryo-electron microscopy (TR-cryo-EM) to capture a molecular movie of the catalytic reaction of NendoU. After the establishment of micro/nano-crystallization conditions for NendoU, mix-and-inject TR-SFX studies will be performed at an x-ray free electron laser (XFEL) to capture snapshots of key reaction steps, including substrate binding as well as the catalytic reaction. Secondly, the mix-and-inject technology developed for Tr-SFX, will be adapted for TR-cryo-EM, which complement the TR-SFX study by unravelling the later time points in catalysis, including product release and the steady state. These time-resolved studies of NendoU will unravel the structure of the transition state and may provide a basis for development of novel drugs for severe coronavirus infections or investigations of the role of NendoU in suppression of the immune response. This RAPID award to Arizona State University is made by the Division of Biological Infrastructure 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.