Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics.

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.

Developments in single-molecule and single-particle fluorescence-based approaches for studying viral envelope glycoprotein dynamics and membrane fusion.

Fusion of viral and cellular membranes is an essential step in the entry pathway of all enveloped viruses. This is a dynamic and multistep process, which has been extensively studied, resulting in the endpoints of the reaction being firmly established, and many essential cellular factors identified. What remains is to elucidate the dynamic events that underlie this process, including the order and timing of glycoprotein conformational changes, receptor-binding events, and movement of the glycoprotein on the surface of the virion. Due to the inherently asynchronous nature of these dynamics, there has been an increased focus on the study of single virions and single molecules. These techniques provide researchers the high precision and resolution necessary to bridge the gaps in our understanding of viral membrane fusion. This review highlights the advancement of single-molecule and single-particle fluorescence-based techniques, with a specific focus on how these techniques have been used to study the dynamic nature of the viral fusion pathway.