INNER BUT NOT OUTER-MEMBRANE LEAFLETS CONTROL THE TRANSITION FROM GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED INFLUENZA HEMAGGLUTININ-INDUCED HEMIFUSION TO FULL FUSION

Cells that express wild-type influenza hemagglutinin (HA) fully fuse t o RBCs, while cells that express the HA-ectodomain anchored to membran es by glycosylphosphatidylinositol, rather than by a transmembrane dom ain, only hemifuse to RBCs. Amphipaths were inserted into inner and ou ter membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer le aflets, amphipaths did not promote the transition, independent of whet her the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In co ntrast, when incorporated into inner leaflets, positive curvature agen ts led to full fusion. This suggests that fusion is completed when a l ipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion condit ions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is t he same pattern of dye spread as in stable hemifusion, for this ''stun ted'' fusion, lower concentrations of amphipaths in inner leaflets wer e required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, th e transmembrane domain causes pore formation by conferring positive sp ontaneous curvature to leaflets of the hemifusion diaphragm.