MEMBRANE ASYMMETRY IS MAINTAINED DURING INFLUENZA-INDUCED FUSION

We have investigated the influence of influenza-induced membrane fusio n on the transverse asymmetry of the viral and target membranes. Large unilamellar vesicles containing headgroup-labeled fluorescent phospho lipid analogues in both leaflets of the membrane were treated with pho spholipase D, converting all outer membrane phospholipids to phosphati dic acid and leading to the release of the fluorescent label from the outside leaflet. After fusion of virus with these liposomes, addition of the enzyme to the fusion product did not release fluorescent label again, indicating that the phospholipid analogues from the inner leafl et of the membranes had not appeared on the outer leaflet. Moreover, t he integral membrane protein hemagglutinin, which is present on the ou ter leaflet of the viral membrane, was quantitatively digested with pr otease after fusion, indicating that hemagglutinin remained on the out er leaflet of the fusion product. Therefore, there is no merger of the inner with outer leaflets of the viral or the liposomal membrane duri ng fusion, and transverse membrane asymmetry is maintained.