EFFECTS OF SPONTANEOUS BILAYER CURVATURE ON INFLUENZA VIRUS-MEDIATED FUSION PORES

Cells expressing the hemagglutinin protein of influenza virus were fus ed to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whe ther spontaneous curvature of each monolayer of a tar-get membrane aff ects the growth of fusion pores. R18 and DiI lowered the transition te mperatures for formation of an inverted hexagonal phase, indicating th at these probes facilitate the formation of negative curvature structu res. The probes are known to translocate from one monolayer of a bilay er membrane to the other in a voltage-dependent manner. The spontaneou s curvature of the cis monolayer (facing the cells) or the trans monol ayer could therefore be made more negative through control of the pola rity of voltage across the planar membrane. Electrical admittance meas urements showed that the open times of flickering fusion pores were sh orter when probes were in trans monolayers and longer when in cis mono layers compared with times when probe was symmetrically distributed. O pen times were the same for probe symmetrically distributed as when pr obes were not present. This, open times were a function of the asymmet ry of the spontaneous curvature between the trans and cls monolayers. Enriching the cis monolayer with a negative curvature probe reduced th e probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysopho sphatidylcholine was placed in trans leaflets of planar membranes, clo sing of fusion pores was rare. The effects of the negative and positiv e spontaneous curvature probes do not support the hypothesis that a fl ickering pore closes fr-om an open state within a hemifusion diaphragm (essentially a ''flat'' structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-d imensional hourglass shape from which the pore flickers shut.