Fig. 2.

Cell-cell fusion assay. BSR-T7 cells in 96-well plates were infected with vTF7-3 at an MOI of 3 and transfected with 0.02 μg/well of VSIV G. At 5 h posttransfection, the VSIV G-expressing cells were overlaid with cells containing a luciferase plasmid, which requires T7 RNA polymerase for expression. Low-pH fusion buffer was added to cells for 2 min to allow for conformational changes in G, thereby producing fusion. Cells were allowed to recover for 1 h, and luciferase levels were analyzed with a luminometer. (A) Wild-type and altered VSIV G proteins were expressed independently; however, only wild-type G was capable of promoting cell-cell fusion. (B) Wild-type VSIV G was combined with an equal molar ratio of fusion-defective VSIV G proteins. Expression of fusion-defective proteins along with the wild type poisons the wild-type fusion reaction. (C) Wild-type VSIV G was combined with various molar ratios of A117F. (D) VSNJV G protein was expressed in equal molar ratios with VSIV fusion-defective proteins. Only Y73A is shown, but W72A, Y116A, and A117F behaved similarly. All experiments were performed four times, and error bars represent the standard deviations between experiments.