Conformational dynamics of viral fusion proteins at single-molecule resolution.

Abstract

The dynamic events that occur during viral membrane fusion during entry into cells have evaded elucidation. Envelope glycoproteins reside on the surface of viral particles, recognize cellular receptors, and promote the fusion of viral and cellular membranes. We have developed multiple single-molecule Förster resonance energy transfer (smFRET) assays to visualize the conformational changes in the Ebola virus envelope glycoprotein (GP) in its native membranous environment on the surface of viral particles. In parallel experiments, we have used fluorescence correlation spectroscopy (FCS) to correlate GP conformation with the ability to interact with a target membrane. Previous studies have identified proteolytic cleavage of GP by host proteases, binding to a cellular receptor, and the chemical environment of the late endosome as being critical during fusion. But the molecular mechanisms by which these events and variables trigger the necessary conformational changes in GP are not known. As a result, a complete and specific model of Ebola fusion, which integrates host factors, environmental conditions, and GP conformational changes currently does not exist. Here, we sought to specify the conformational changes undergone by GP during exposure to the factors and cues that Ebola encounters in the late endosome. Our results support a working model in which acidic pH and Ca2+ stabilize a GP conformation capable of binding a membrane. Receptor binding stabilizes an extended intermediate, which is sufficient for lipid mixing, but insufficient to trigger formation of the post-fusion GP conformation. Additional endosomal cues are currently being investigated for a potential role in promoting conversion to the post-fusion state.