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

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Principal Investigator: Mario  Borgnia
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2024
Award: $727,531
Funding agency: National Institute of Environmental Health Sciences

The goal of this project is to understand the molecular mechanisms involved in the infection of mammalian cells by enveloped viruses. Using cryogenic electron microscopy (cryo-EM) we have established workflows to determine the structure of the molecular machines involved in these processes. We solve the structure of purified macromolecular complexes using single particle analysis cryo-EM (SVA). To solve structures in pleiomorphic environments, we resort to cryo-electron tomography (cryo-ET) combined with sub-volume averaging techniques (SVA). The later approach allows for the determination of structures in their biological context in thin specimens such as viral particles. We have previously developed novel approaches to increase the through of cryo-ET (Bouvette J et al. Nat Commun. 2021 Mar 30;12(1):1957). A focused ion beam integrated into a scanning electron microscope allows us to excise regions of interest in cryogenically preserved biological materials. This cryo-FIBSEM method allows us to study macromolecular complexes in samples such as virus infected cells which are otherwise not amenable to cryo-electron microscopy due to their thickness. Thus, we have deployed cryo-FIBSEM for the identification and isolation of regions of interest to be imaged using high resolution cryo-ET. 
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.

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