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.
Highlights
Ebola virus (EBOV) disease outbreaks in West and Sub-Saharan Africa have occurred since the emergence of the virus in 1976 [1]
As the sole surface antigen of EBOV, GP is the target of host neutralizing antibodies, which may function to inhibit conformational changes required for membrane fusion [10]
We sought to probe movement of GP1 with respect to GP2, as well as movement of the viral fusion loop that is proximal to the N terminus of GP2
Summary
Ebola virus (EBOV) disease outbreaks in West and Sub-Saharan Africa have occurred since the emergence of the virus in 1976 [1]. Following attachment to the cell surface, EBOV is trafficked to late endosomes where GP1 is proteolytically cleaved by host cathepsins to remove the mucin-like domain (muc) and the glycan cap, forming GPCL [3,4]. This cleavage event exposes the binding site for the host receptor for EBOV, Niemann-Pick C1 protein (NPC1). Binding of GPCL to the luminal domain C of NPC1 (NPC1-C) is necessary, but not sufficient, to trigger GP-mediated fusion of the viral and endosomal membranes [5,6,7,8,9]. As the sole surface antigen of EBOV, GP is the target of host neutralizing antibodies (nAbs), which may function to inhibit conformational changes required for membrane fusion [10]
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