Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7–20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.


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