HIV-1 Env-mediated membrane fusion monitored by fluorescence microscopy of syncytium formation between Clone69T1RevEnv and SupT1 cells
Nejat Düzgüneş: 0000-0001-6159-1391
Biophysical Society 54th Annual Meeting
San Francisco, CA
Feb. 20-24, 2010
Date of Presentation
Objectives: HIV-1 initiates infection by fusing with CD4+ lymphocytes or macrophages, mediated by the envelope protein Env (gp120/gp41). We developed a new HIV fusion assay using fluorescently labeled Clone69T1RevEnv cells expressing Env, and CD4+ SupT1 cells. We examined whether known inhibitors of HIV-1 infection, including peptides, lectins and antibodies inhibit membrane fusion in this system.Methods: Clone69TRevEnv (“HIV-Env”) cells were maintained in DMEM with heat-inactivated fetal bovine serum (FBS), L-glutamine, geneticin, hygromycin B and tetracycline. Env expression was induced by removing tetracycline. Cells were plated in 48-well plates for 24 h and labeled with Calcein-AM Green. SupT1 cells, maintained in RPMI 1640 with 10% FBS, penicillin, streptomycin and L-glutamine, were labeled with CellTrace(tm) Calcein red-orange. The SupT1 cells were incubated with adherent HIV-Env cells, with or without the inhibitors, for 3 h, and observed under a Nikon fluorescence microscope. Syncytia formation resulted in orange fluorescence. Results: Hippeastrum hybrid (Amaryllis) agglutinin (HHA), Galanthus nivalis (Snowdrop) agglutinin (GNA), and the peptide Enfuvirtide inhibited membrane fusion at 1 μg/ml. Anti-gp120 (IgG1B12, m14 IgG, and 2G12), and anti-gp41 (2F5 and 4E10) antibodies that inhibit HIV-1 infection, had little or no inhibitory effect on cell-cell fusion. Conclusion: Fluorescently labeled HIV-Env and SupT1 cells can be used to monitor HIV Env-mediated fusion as the fused cells change color. This assay can be adapted to screen novel inhibitors of membrane fusion in high-throughput assays. Our observation that antibodies that inhibit HIV infection do not inhibit syncytium formation, suggests that the mechanisms of cell-cell and virus-cell membrane fusion may be different. This observation also raises the possibility that the antibodies may not be able to inhibit the spread of viral genetic material from infected cells to uninfected cells.
HIV-1 Env-mediated membrane fusion monitored by fluorescence microscopy of syncytium formation between Clone69T1RevEnv and SupT1 cells.
Paper presented at Biophysical Society 54th Annual Meeting in San Francisco, CA.