Epitope dynamics and functional analysis of viral envelope glycoprotein by cryo-electron tomography and single particle cryo-EM

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We have established high-throughput structure determination workflows using single particle analysis cryo-EM (SPA) on variants of the SARS-CoV-2 S protein ectodomain. These workflows allow us to solve the structure of macromolecular complexes of the spike at near atomic resolution with less than 24 hours of combined data collection and image processing. We also developed the first framework for automated evaluation of cryo-EM specimens using machine learning (Bouvette et. Al, eLife 2022). In collaboration with Dr. Robert Petrovich at the Protein Expression and Purification Facility, we established a pipeline to determine the structure of S-protein ectodomain from different variants of concern. Over the past three years we have used this method to solve the structure of the spike in complex with a variety of ligands in several parallel projects. In FY-2022 we published the results of two of these projects (Hong et al. PNAS 2022 and Fu et al. Plos One 2022) resulting from collaborations with the groups of Dr. Mitchell Ho at NCI and Dr. Matthew Hall at NCATs. We have deployed a cryo-electron tomography (cryo-ET) and sub-volume averaging (SVA) pipeline to determine the structure of full length (FL) S-protein and its complexes in the context of the viral envelope. In collaboration with Dr. Alberto Bartesaghi at Duke we are establishing modernized cryo-ET/SVA workflows that make use of the improved quality of DED data and incorporate novel image processing techniques to obtain high-resolution tomographic reconstructions, identify objects of interest in a crowded environment and determine their near-atomic resolution structure (Bouvette et al. Nat. Comm. 2021). In collaboration with Dr. Eric Freed at NCI and Dr. Negin Martin at the NIEHS Viral Vector Core we have established BSL-2 compatible pseudotyped viral systems for expression of wild type and mutant forms of type I fusion proteins from SARS-CoV-2 and HIV. These models will help shed light on aspects of the cellular pathogenesis of AIDS and COVID-19. We are currently using these systems in several collaborative projects aimed to a) characterize epitopes on the surface of the spike, b) map conformational changes along the maturation process, and c) map the interaction of S1/S2 with intracellular receptors and epithelial macromolecules. A model for the mechanism of fusion mediated by these proteins has been proposed based on their structures in the prefusion and post fusion states, and on biophysical data at much lower resolution. The model postulates conformational intermediates which are yet to be confirmed experimentally. Their elucidation will require the development of structural techniques with sufficient temporal resolution to capture intermediate snapshots. In collaboration with Dr. Tony Huang at Duke University, we are developing novel approaches to this problem. In addition to shedding light on the mechanism of fusion, this instrumentation will provide tools for the structural dissection of a wide variety of dynamic processes. Structural characterization of other stages of viral replication will require access to structural determination in situ. We are deploying a cryogenic focused ion beam scanning electron microscope. We will combine this new capability with our high throughput tomographic structure determination pipeline to characterize macromolecular complexes in the context of the cell.