MEMBRANE-FUSION OF SEMLIKI FOREST VIRUS REQUIRES SPHINGOLIPIDS IN THETARGET MEMBRANE

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis , fusion of the viral envelope occurring subsequently from within acid ic endosomes. Fusion of SFV has been demonstrated before to be strictl y dependent on the presence of cholesterol in the target membrane. Her e, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate tha t low-pa-induced fusion of SFV with cholesterol-containing liposomal m odel membranes requires the presence of sphingomyelin or other sphingo lipids in the target membrane. The minimal molecular characteristics e ssential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, ch olesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipi ds with cholesterol is unlikely to be important for the induction of S FV-liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half- maximal fusion (1-2 mole%) suggest that sphingolipids do not play a st ructural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.