Author ORCiD
Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Seth A. Darst
Additional Thesis Advisor
Elizabeth Campbell
Keywords
cryo-electron microscopy (Cryo-EM), NiRAN domain, coronaviruses, replication-transcription complexes
Abstract
The COVID-19 pandemic was a dramatically disruptive event, wreaking devastation across the globe and radically impacting nearly every facet of human life. For the first time, a significant amount of global money and attention was directed at coronavirus (CoV) research, propelling the field to the forefront of biomedical research. This allowed for the rapid development of a suite of vaccines and antivirals targeting SARS-CoV-2 that aided in ameliorating the effects of the pandemic. SARS-CoV-2 was the third CoV zoonosis of the 21st century and is now endemic. Global understanding of the threats posed by CoVs circulating among animals in the wild has increased, powering the urgency for new research into CoV biology. We study CoV replicase enzymes to shed light on the mechanisms underpinning viral replication and gene expression in the hope to elucidate biology conserved across viruses and better define therapeutic targets. The enzymatic activity of the SARS-CoV-2 Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain is essential for viral propagation, with three distinct activities associated with modification of the nsp9 N terminus, NMPylation, RNAylation, and deRNAylation/capping via a GDP-polyribonucleotidyltransferase reaction. The latter two activities comprise an unconventional mechanism for initiating viral RNA 5′ cap formation, while the role of NMPylation is unclear. Prior to this work, the structural mechanisms for these diverse enzymatic activities have not been properly delineated, nor have any drugs targeting this domain been clinically approved. In our initial work, we determined high-resolution cryo-electron microscopy (cryo-EM) structures of catalytic intermediates for the NMPylation and deRNAylation/capping reactions with their preferred substrates. These data revealed diverse nucleotide binding poses and divalent metal ion coordination sites within the NiRAN domain that promote its repertoire of activities. The deRNAylation/capping structure explained why GDP is the preferred substrate for the capping reaction over GTP. Altogether, these findings enhanced our understanding of the promiscuous activity of the CoV NiRAN domain and provided an accurate structural platform for drug development. Following this study, we sought to address a flawed study published in Cell claiming to have defined the structural basis of GTP-mediated capping by the NiRAN domain. We demonstrated that their model is not supported by their cryo-EM data and is incompatible with fundamental chemical principles. We discuss our failures, and those of the original authors, at reprocessing the data and identifying an authentic intermediate, clarifying that the mechanism remains unknown. Correcting this model was critical to restoring focus and reorienting the field toward supported models of CoV mRNA capping. Additionally, we present work that details a pipeline for evaluating inhibitors against the NiRAN domain. This pipeline includes two high-throughput assays to monitor NiRAN NMPylation and capping activities, respectively, as well as a means of detecting binding to the NiRAN domain. We reveal early data suggesting the existence of a druggable cryptic pocket in the NiRAN domain and present a path to structurally characterizing the pocket. Finally, we also share preliminary efforts at isolating the pore complex that gates the CoV replication organelle, with the ultimate goal of cryo-EM analysis, work that has the potential to address a host of unknowns in CoV replication.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Small, Gabriel I., "Structural and Functional Studies of the SARS-CoV-2 NiRAN Domain" (2026). Student Theses and Dissertations. 844.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/844
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy