Fig. 4
HSV-1 gD and gB do not significantly affect the processing of HIV-1 Gag and gp160. 293 cells were co-transfected with empty pcDNA3.1(+) vector or one expressing gD and pNL4-3. At 24 h, cells were starved in medium lacking methionine/cysteine for 2 h followed by radiolabeling cultures with 35S-methionine/cysteine. The radiolabel was removed and washed three times in medium containing 100X methionine/cysteine and chased in the same medium for 0, 1, 3, and 6 h. The culture medium was harvested and cell lysates prepared as described in the Materials and Methods. HIV-1 Env and Gag proteins were immunoprecipitated using the HIV-1 antibodies and HSV-1 gD was immunoprecipitated with an appropriate monoclonal antibody. The immunoprecipitates were collected on protein-A-Sepharose beads, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS gels and visualized using standard radiographic techniques. a, b HIV-1 proteins immunoprecipitated from the cell lysates (a) and culture medium (b) of cells co-transfected with pcDNA3.1(+) and pNL4-3. c, d HIV-1 proteins immunoprecipitated from the cell lysates (c) and culture medium (d) of cells co-transfected with a vector expressing gD and pNL4-3. e, f HSV-1 gD protein immunoprecipitated from cell lysates (e) and culture medium (f) of cells co-transfected with a vector expressing gD and pNL4-3