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

JM Smit, R Bittman, J Wilschut*

*Corresponding author for this work

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    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 Dr 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 El homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the El trimer represents the fusion-active conformation of the viral spike.

    Original languageEnglish
    Pages (from-to)8476-8484
    Number of pages9
    JournalJournal of Virology
    Volume73
    Issue number10
    Publication statusPublished - Oct-1999

    Keywords

    • SEMLIKI-FOREST-VIRUS
    • VESICULAR STOMATITIS-VIRUS
    • MEMBRANE-FUSION
    • SPIKE PROTEIN
    • CONFORMATIONAL-CHANGES
    • NUCLEOTIDE-SEQUENCE
    • TARGET MEMBRANE
    • INFLUENZA-VIRUS
    • MODEL SYSTEM
    • BHK CELLS

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