Membrane asymmetry is maintained during influenza-induced fusion

We have investigated the influence of influenza-induced membrane fusion on the transverse asymmetry of the viral and target membranes. Large unilamellar vesicles containing headgroup-labeled fluorescent phospholipid analogues in both leaflets of the membrane were treated with phospholipase D, converting all outer membrane phospholipids to phosphatidic 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 leaflet of the membranes had not appeared on the outer leaflet. Moreover, the integral membrane protein hemagglutinin, which is present on the outer leaflet of the viral membrane, was quantitatively digested with protease after fusion, indicating that hemagglutinin remained on the outer leaflet of the fusion product. Therefore, there is no merger of the inner with outer leaflets of the viral or the liposomal membrane during fusion, and transverse membrane asymmetry is maintained.