SUMMARY The increasing frequency and severity of Ebola outbreaks demands an expanded repertoire of treatments and preventative measures. Answering this unmet need will require a deeper understanding of the molecular mechanisms underlying filovirus replication. In particular, the dynamic events that occur during filovirus envelope glycoprotein (GP)-mediated membrane fusion during entry into cells have evaded elucidation for decades. Previous studies have identified proteolytic cleavage of GP, receptor binding, and the chemical environment of the late endosome as being critical. But the molecular mechanisms by which these events and variables promote GP-mediated membrane fusion are not known. As a result, a complete and specific model of filovirus fusion, which integrates host factors, environmental conditions, and GP conformational changes currently does not exist. Therefore, filovirus entry continues to be an unutilized target for inhibitors. Our long-term goal is to develop a complete mechanistic model of GP-mediated membrane fusion. Our recent publications, in which we demonstrate the power of single-molecule fluorescence methods in elucidating the conformational dynamics of EBOV GP on the surface of virions, demonstrate our initial efforts toward this end. Here we aim to build on this success by proposing a multidisciplinary study involving virological, cellular, structural, and biophysical methodologies to elucidate the dynamics and mechanisms of GPs from multiple filoviruses. We will characterize the mechanisms by which conformational changes, host factors, and environmental variables facilitate filovirus membrane fusion and entry into cells. Completion of the proposed research will provide mechanistic insights into filovirus entry and the viral and host targets that could be exploited with novel therapies and immunogens.