Allosteric remodelling of the Ebola virus glycoprotein underlies differences in entry mechanisms across species

During Ebolavirus transit through the endo-lysosomal pathway, the glycoprotein GP undergoes a series of conformational rearrangements upon cathepsin cleavage and NPC1 receptor binding, culminating in host-virus membrane fusion. While these rearrangements underpin viral entry, their molecular mechanisms remain poorly understood. Here, we combine hydrogen/deuterium-exchange mass spectrometry and mass photometry to resolve structural dynamic and energetic transitions of GP of two major species, Zaire (EBOV) and Sudan (SUDV). We describe the allosteric axes that govern reorganization of the cleavage sites, opening of the receptor binding cavity and priming for fusion upon NPC1 engagement. We show that GP cathepsin cleavage and receptor binding exhibit different mechanisms and kinetics across species, and are allosterically coupled in a species-specific manner. We reveal that fusion priming is optimized via distinct routes in EBOV and SUDV GP. These data suggest that GP structural dynamics and allosteric remodelling underlie differences in entry mechanisms across Ebolavirus species.