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

Abstract

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.

New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

“…Thus, the authors conclude that low pH and endosomal Ca 2+ act to prime GP for receptor binding following removal of the glycan cap by promoting transition to the receptor-bindingcompetent intermediate state while maintaining reversibility. Similarly, as observed by Durham et al, removal of the glycan cap resulted in a repositioning of GP1 with respect to GP2 and increased mobility in the GP2 fusion loop [104,105]. Together these results indicate that removal of the glycan cap, low pH, and Ca 2+ act synergistically to promote an intermediate state primed for receptor Additional factors implicated in Ebola entry and GP-mediated fusion include endosomal pH and exposure to Ca 2+ [112].…”

“…In this study, the donor and acceptor fluorophores were positioned on GP1 and GP2 so that the movement of GP1 with respect to GP2 could be monitored. The authors suggest that removal of the glycan cap results in a repositioning of GP1 that favors NPC1 binding and relieves conformational restrictions on GP2, conferring increased flexibility and mobility to GP2 and the fusion loop [104,105]. Thus, the reversible structural changes and dynamics observed here likely correspond to a repositioning of GP1 with respect to GP2 where GP1 does not dissociate from GP2 upon receptor binding.…”

“…As noted above, in contrast to influenza HA, other type I viral fusion systems present a more complex set of activation factors. Das et al and Durham et al recently sought to characterize the intrinsic structural dynamics and dynamic conformational changes that occur in another type I fusion protein, the Ebola virus GP fusion glycoprotein, resulting from a set of activating factors [104,105]. Once internalized into cells by macropinocytosis, the low pH conditions of the endosome activate cellular proteases that cleave and remove the heavily glycosylated mucin-like domain and glycan cap from GP [104,[106][107][108][109].…”

“…Atomic resolution structures of the pre-fusion GP ectodomain without the mucin-like and transmembrane domains (GP∆TM) depict GP∆TM in a single, static conformation [74,106]. However, by sm-FRET Durham et al observed GP∆TM to be highly dynamic and capable of reversibly interconverting between three distinct states, with the dominant high-FRET state corresponding to the pre-fusion state depicted in the crystal structure [105]. Similar dynamics were observed for GP without the mucin-like domain (GP∆muc) when presented on the surface of pseudovirus particles ( Figure 3A).…”

“…Thus, the reversible structural changes and dynamics observed here likely correspond to a repositioning of GP1 with respect to GP2 where GP1 does not dissociate from GP2 upon receptor binding. It is important to note that while removal of the glycan cap and NPC1 binding are necessary, they alone are not sufficient to trigger GP-mediated membrane fusion [104,105]. Additional factors implicated in Ebola entry and GP-mediated fusion include endosomal pH and exposure to Ca 2+ [112].…”