Chao, LukeKlein, DarylSchmidt, AaronPena, JenniferHarrison, Stephen2015-02-022014Chao, Luke H, Daryl E Klein, Aaron G Schmidt, Jennifer M Peña, and Stephen C Harrison. 2014. “Sequential conformational rearrangements in flavivirus membrane fusion.” eLife 3 (1): e04389. doi:10.7554/eLife.04389. http://dx.doi.org/10.7554/eLife.04389.2050-084Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:13890587The West Nile Virus (WNV) envelope protein, E, promotes membrane fusion during viral cell entry by undergoing a low-pH triggered conformational reorganization. We have examined the mechanism of WNV fusion and sought evidence for potential intermediates during the conformational transition by following hemifusion of WNV virus-like particles (VLPs) in a single particle format. We have introduced specific mutations into E, to relate their influence on fusion kinetics to structural features of the protein. At the level of individual E subunits, trimer formation and membrane engagement of the threefold clustered fusion loops are rate-limiting. Hemifusion requires at least two adjacent trimers. Simulation of the kinetics indicates that availability of competent monomers within the contact zone between virus and target membrane makes trimerization a bottleneck in hemifusion. We discuss the implications of the model we have derived for mechanisms of membrane fusion in other contexts. DOI: http://dx.doi.org/10.7554/eLife.04389.001en-USmembrane fusionflavivirusWest Nile virussingle particlekineticsconformational changevirusesJournal Article2015-02-0210.7554/eLife.04389