SPHINGOLIPIDS ACTIVATE MEMBRANE-FUSION OF SEMLIKI-FOREST-VIRUS IN A STEREOSPECIFIC MANNER

The alphavirus Semliki Forest virus (SFV) enters cells through recepto r-mediated endocytosis. Subsequently, triggered by the acid pH in endo somes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presenc e of cholesterol in the target membrane. Recently, we have demonstrate d that fusion of SFV also requires sphingolipids [Nieva, J. L., Bron, R., Corver, J., & Wilschut, J. (1994) EMBO J. 13, 2797-2804]. In the p resent paper, we show that the activation of low-pH-dependent fusion o f SFV by sphingolipids is a stereospecific process. Pyrene-labeled SFV fused rapidly and extensively with liposomes consisting of a mixture of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, sup plemented with low concentrations of D-erythro-ceramide, representing the naturally occurring sphingolipid stereoisomer. Fusion was assessed by a decrease in the pyrene excimer fluorescence. L-erythro-, D-threo -, and L-threo-ceramide did not support fusion of the virus. Similar r esults were obtained with the corresponding sphingomyelin stereoisomer s, The stereospecificity of SFV fusion activation was confirmed by usi ng an assay based on degradation of the viral capsid protein by trypsi n encapsulated in the target liposomes. Fusion mediated by D-erythro-c eramide was not affected by the additional presence in the target lipo somes of ceramide stereoisomers incapable of fusion activation. Bindin g of the virus to the liposomes, as assessed by flotation on sucrose d ensity gradients, was not dependent on the presence of fusion-competen t or fusion-incompetent sphingolipids in the liposomes, The results of this study support the notion that a stereospecific interaction of th e viral fusion protein with D-erythro sphingolipids in the target memb rane represents an essential step in the activation of the fusion capa city of SFV.