Low-pH-dependent fusion of sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes

There is controversy as to whether the cell entry mechanism of Sindbis viru s (SIN) involves direct fusion of the viral envelope with the plasma membra ne at neutral pH Dr uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the me mbrane fusion activity of SIN in a liposomal model system. Fusion was follo wed fluorometrically by monitoring the dilution of pyrene-labeled lipids fr om biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of de gradation of the viral core protein by trypsin encapsulated in the liposome s, SIN fused efficiently with receptor-free liposomes, consisting of phosph olipids and cholesterol, indicating that receptor interaction is not a mech anistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a ce ll entry mechanism of SIN involving fusion from within acidic endosomes. Un der optimal conditions, 60 to 85% of the virus fused, depending on the assa y used, corresponding to all of the virus bound to the liposomes as assesse d in a direct binding assay. Preincubation of the virus alone at pH 5.0 res ulted in a rapid loss of fusion capacity. Fusion of SIN required the presen ce of both cholesterol and sphingolipid in the target liposomes, cholestero l being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions , the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, w ith formation of a trypsin-resistant El homotrimer, which kinetically prece ded the fusion reaction, thus suggesting that the El trimer represents the fusion-active conformation of the viral spike.