Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

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.This work was supported by a Senior Research Fellowship from the Wellcome Trust, grant number 101908/Z/13/Z, to Y.M.; grant R01 GM102869 from the National Institutes of Health (NIH) to Y.M.; NIH grant NS079955 to E.R.; NIH grant T32 GM08283 to J.B.N.; and NIH grant T32 GM007223 to D.C.D. We thank Michel Ledizet, Martin Mattessich and Nathalie Bonafé (L2 Diagnostics, LLC) for helpful discussions.This is the author accepted manuscript. The final version is available from the Society for General Microbiology via http://dx.doi.org/10.1099/vir.0.00011