Sphingolipid-dependent fusion of Semliki Forest virus with cholesterol-containing liposomes requires both the 3-hydroxyl group and the double bond of the sphingolipid backbone.

Low-pH-induced membrane fusion of Semliki Forest virus (SFV) in a model system is mediated by sphingolipids in the target membrane; ceramide is the sphingolipid minimally required (J. L. Nieva, R. Bron, J. Corver, and J. Wilschut, EMBO J. 13:2797-2804, 1994). Here, using various ceramide analogs, we demonstrate that sphingolipid-dependent fusion of SFV with cholesterol-containing liposomes exhibits remarkable molecular specificity, the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone being critical for the sphingolipid to mediate the process. This observation supports the notion that sphingolipids act as a cofactor in SFV fusion, interacting directly with the viral fusion protein to induce its ultimate fusion-active conformation.