GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.
Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA-expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.
Melikyan
G B
GB
Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612, USA.
White
J M
JM
Cohen
F S
FS
D000818:Animals; D016466:CHO Cells / Q000502:physiology; D002459:Cell Fusion / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology*; D004396:Coloring Agents / Q000493:pharmacokinetics; D006224:Cricetinae; D004594:Electrophysiology; D004912:Erythrocytes / Q000502:physiology*; D004996:Ethidium; D017261:Glycosylphosphatidylinositols / Q000502:physiology*; D006389:Hemagglutinins, Viral / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D050356:Lipid Metabolism; D009975:Orthomyxoviridae / Q000737:chemistry; D009995:Osmosis / Q000502:physiology; D018272:Porins / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000502:physiology
D004396:Coloring Agents; D017261:Glycosylphosphatidylinositols; D006389:Hemagglutinins, Viral; D008051:Lipid Bilayers; D018272:Porins; D014760:Viral Fusion Proteins; D004996:Ethidium