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.
1Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA.
Danillo L A
DL
Espósito
1Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA.
Jennifer B
JB
Nguyen
1Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA.
David C
DC
DeWitt
1Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA 2Department of Physics, Yale University, New Haven, CT 06520, USA.
Elizabeth
E
Rhoades
1Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA.
Yorgo
Y
Modis
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D055598:Chemical Phenomena; D018349:Encephalitis Viruses, Japanese / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D008561:Membrane Fusion; D013696:Temperature; D013997:Time Factors; D022701:Virosomes / Q000378:metabolism; D014902:West Nile virus / Q000502:physiology*
D008055:Lipids; D022701:Virosomes