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.
