FIG 3 .

The KGD motif is involved in interaction of gH/gL with gp42. (A) The gH-AAA/gL mutant has decreased association with gp42. CHO-K1 cells seeded in six-well plates were transfected with control, gH/HA-gL, or mutant gH-AAA/HA-gL. Twenty-four hours posttransfection, the cells were washed twice with ice-cold PBS and incubated with media containing Flag-tagged gp42 (isolated 24 h after transfection) for 1 h at 4°C. The cells were then washed with ice-cold PBS four times and lysed with 200 µl of 1× SDS lysis buffer. Proteins were separated on 10% SDS-polyacrylamide gels. Western blot analyses were performed using the polyclonal anti-Flag antibody (F7425) at 1:1,000 to detect the Flag-gp42 that bound to the transfected cells. Anti-GAPDH and anti-HA (gL-HA) were used as loading controls. The data are representative of three independent experiments. (B) Soluble gp42 can decrease the binding of gH/gL with epithelial cells. CHO-K1 cells were transiently transfected with control, gH/gL, or gH-AAA/gL. Twenty-four hours posttransfection, 2 × 10⁶ cells were collected, and soluble gH/gL or gH-AAA/gL was prepared. Soluble gp42 was added as described in Materials and Methods. Binding of gH/L or gH-AAA/gL to HEK 293 cells was measured following overlay of HEK 293 cells with soluble gH/gL or gH-AAA/gL with or without soluble gp42 for 4 h at 4°C followed by detection of gH binding by CELISA with gH/gL antibody E1D1. The data are means plus SD from three independent experiments.