Structural Characterization of SARS-CoV-2 Genome Organization by NMR

The goal of this project is to structurally characterize the nucleocapsid (N)-mediated packaging of the genome of the SARS-CoV-2 virus, the causative agent of the deadly COVID-19 pandemic. The proposed investigations and the general methodological framework to be developed will have broad applications since they will provide 1) biological insights that open new strategies for interfering with viral genome packaging, and 2) integrative magnetic resonance-based protocols that can be used by other practitioners (biochemists, biophysicists, molecular biologists). As such, the proposed research will impact several areas of science, including structural biology, biochemistry, physical chemistry, magnetic resonance spectroscopy, biotechnology and pharmacology. The proposed innovative research program will provide unique training for students at all levels in state-of-the-art experimental methods. Importantly, an educational program within this project will include embedding opportunities for members of the laboratories of the PI, Co-PI and the industrial collaborator that will greatly enhance the training of the STEM workforce. The goal of this project is to structurally characterize how the nucleocapsid (N) packages the genome in the SARS-CoV-2 virus, by integrating experimental magic angle spinning (MAS) and dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) methods. This new methodological framework, established through the proposed research, will overcome the current challenges in studying complex heterogeneous viral genome ribonuclear particles that exhibit different degrees of disorder and dynamics. The proposed research will provide atomic-level knowledge on the structure and dynamics of the SARS-CoV-2 N protein as well as the structural organization of the N protein in assemblies with genomic RNA. Employing high magnetic fields together with fast and ultrafast 1H- and 19F- detected MAS and DNP-enhanced MAS NMR will enable studies into very large virus systems, requiring only a fraction of the material used in conventional experiments. The general methodological framework that will be developed will be broadly applicable to investigate packaged genomes of other viruses and can be used by a wide range of practitioners of these methodologies (biochemists, biophysicists, molecular biologists). As such, the planned research will impact several areas of science, including structural biology, biochemistry, physical chemistry, magnetic resonance spectroscopy, biotechnology and pharmacology. 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.