FIG. 8.

Cell-cell fusion assay of Glu 1, Glu 2, Lys 1, and Lys 2. 293T cells were cotransfected with pOS8 and pCB6 vectors expressing Env proteins; 48 h posttransfection, cells were lifted off the dish and split into three samples that were either checked for surface expression of envelope proteins or used in the cell-cell fusion assay. A portion of transfected 293T cells was overlaid onto Tva-expressing 3T3 cells which had been infected with MVA. After 12 h at 37°C, samples were either fixed and stained with X-Gal (A) or lysed and analyzed in the MUG β-galactosidase fluorometric assay (B). Results of the β-galactosidase fluorometric assay are expressed as fluorescence units and represent averages from three replicates; bars show standard deviations. All samples were read in the linear range of fluorescence emission. (C) A portion of transfected 293T cells were biotinylated with NHS-LC-biotin, lysed, and immunoprecipitated with anti-A tail antibody. Samples were analyzed as described in the legend to Fig. 2B. (D) Another subset of transfected 293T cells was incubated on ice with the anti-gp37 antibody in a total volume of 100 μl PBS–2% FCS for 30 min. Cells were washed twice in PBS and then incubated with a fluoresceinated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories) in 100 μl of PBS–2% FCS on ice. After 30 min, cells were washed twice with PBS, resuspended in PBS containing 2% paraformaldehyde, and analyzed on a Becton Dickinson flow cytometer.