Figure 4

pH and cholesterol dependency in cells induced by monomeric-E1-based constructs. (A) Sf21 cells were infected with the indicated recombinant baculoviruses. After 1 dpi, the culture medium was replaced with Sf-900 II SFM (pH = 5.8) containing 2% FCS and 100 μg/ml cholesterol to triggering cell-cell fusion. Syncytial formation was examined under a fluorescence microscope with green channel. The bar represents 50 μm. (B) pH profiles of cell fusion induced by wild type or mutant E1. Sf21 cells were infected with the indicated recombinant baculoviruses. After 1 dpi, the culture medium was replaced with Sf-900 II SFM containing 2% FCS, 100 μg/ml cholesterol and pH levels of 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, and 6.9 as indicated. Syncytial formation was examined under a fluorescence microscope. At least 100 nuclei per field were counted at a 200× magnification. The fusion index was determined using: Number of multiple nuclei cells/number of EGFP positive cells. (C) Cholesterol-dependent profiles. Sf21 cells that passaged at least three times in Sf-900 II SFM were infected with the indicated recombinant baculoviruses. After 1 dpi, the culture medium was replaced with Sf-900 II SFM (pH = 5.8) containing 2% FCS and, various levels of cholesterol as indicated. Syncytial formation was examined under a fluorescence microscope. Approximately 100 nuclei per field were counted at 200× magnification. The fusion index was determined as described above.