HIV-1 envelope glycoprotein-mediated membrane fusion monitored by fluorescence microscopy
Nejat Düzgüneş: 0000-0001-6159-1391
85th General Session of the International Association for Dental Research (IADR) and 36rd Annual Meeting of the American Association for Dental Research (AADR)
New Orleans, LA
March 21-24, 2007
Date of Presentation
Journal of Dental Research
Journal Volume Number
86 (Special issue A)
HIV-1 infects host cells by fusion at the plasma membrane, mediated by the envelope glycoprotein gp120/gp41 (Env). This process can be studied by monitoring syncytia formation between HIV-infected cells and target CD4+ cells. The fusion reaction can also be observed by the accompanying intermixing of aqueous contents or membrane components, using Env-expressing cells containing fluorescent probes. Objectives: We examined whether antibody and peptide inhibitors of viral infection also inhibit Env-mediated cell-cell fusion, and whether Env remains fusion-competent even after prolonged incubation with peptide inhibitors. Methods: We studied the fusion of the B-cell line TF228.1.16 cells expressing gp120/gp41, with HeLa cells expressing CD4, using fluorescence microscopy. HeLa-CD4 cells were loaded with Calcein AM, which is trapped in the cytoplasm following esterase cleavage, and TF228 cells were labeled with CellTracker Calcein Red-Orange. The cells were incubated for 1 h at 25¢ªC to facilitate adhesion, and then at 37¢ªC to initiate fusion, resulting in the formation of orange syncytia. Results: The peptides T20, C34 and N36 were highly inhibitory to fusion in the range 0.2-1 mg/ml. An anti-gp120 antibody (2G12) also inhibited fusion at 1 mg /ml. In contrast, the anti-gp41 antibodies, 1240, 4E10, 2F5, which are known to inhibit viral infection, were not effective in inhibiting fusion. If the peptide inhibitors were removed following a 1 h incubation, fusion resumed, indicating that gp41 remains fusion-potent even after exposure to the inhibitor, and does not undergo an irreversible conformational change following gp120 binding to the receptors and co-receptors on HeLa cells. Conclusions: The fluorescence assay for Env-mediated cell fusion is a convenient system for screening potential inhibitors of HIV-1 infection. However, not all inhibitors of HIV-1 infection inhibit cell-cell fusion in this system. Env remains potentially fusogenic during its interaction with the peptide inhibitors.
HIV-1 envelope glycoprotein-mediated membrane fusion monitored by fluorescence microscopy.
Paper presented at 85th General Session of the International Association for Dental Research (IADR) and 36rd Annual Meeting of the American Association for Dental Research (AADR) in New Orleans, LA.