PMID:10482600

Citation	Smit, JM, Bittman, R and Wilschut, J (1999) Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes. J. Virol. 73:8476-84
Abstract	There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic 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 cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol 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, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.
Links	PubMed PMC112867
Keywords	Animals; Cell Line; Cholesterol/chemistry; Cholesterol/metabolism; Cricetinae; Liposomes; Receptors, Virus; Sindbis Virus/chemistry; Sindbis Virus/physiology; Sphingolipids/chemistry; Sphingolipids/metabolism; Virus Replication
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SINDV:POLS		GO:0098029: icosahedral viral capsid, spike	ECO:0000314:		C		Figure 7.	complete CACAO 6880
SINDV:POLS	involved_in	GO:0061025: membrane fusion	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
SINDV:POLS	part_of	GO:0098029: icosahedral viral capsid, spike	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
SINDV:POLS		GO:0061025: membrane fusion	ECO:0000314:		P		Figure 3.	complete CACAO 6868

References

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