Fig. 5

HSV-1 gD and gB are efficiently incorporated into HIV-1 particles. 293 cells were co-transfected with either empty pcDNA3.1(+) vector, one expressing the HSV-1 gD or HSV-1 gB and pNL4-3. At 30 h, the cells were starved for methionine/cysteine for 2 h and then radiolabeled for with ³⁵S-methionine/cysteine for 16 h. At 48 h post-transfection, the cell culture medium was harvested and subjected to low speed centrifugation to remove cellular debris and virus pelleted through a 20% sucrose cushion using ultracentrifugation. The cells were lysed in RIPA buffer on ice, centrifuged to remove the nuclei. The resulting supernatant was transferred to a new microfuge tube. The medium, cell lysates, and virus pellet  were used in immunoprecipitation analysis using anti-HIV-1 antibodies (to immunoprecipitate Env and Gag proteins) and appropriate monoclonal antibodies to immunoprecipitate HSV-1 gD and gB. The immunoprecipitates were collected on protein-A-Sepharose beads, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS-PAGE and visualized using standard radiographic techniques. a HIV-1 and gD proteins immunoprecipitated from cell lysates prepared from cells co-transfected with either empty vector and pNL4-3 or a vector expressing gD and pNL4-3. b Cells were co-transfected with either empty vector and pNL4-3 or a vector expressing gD and pNL4-3 followed by immunoprecipitation of HIV-1 and gD proteins from the culture medium and pellet after ultracentrifugation. c HIV-1 and gB proteins immunoprecipitated from cell lysates prepared from cells co-transfected with either empty vector and pNL4-3 or a vector expressing gB and pNL4-3. d Cells were co-transfected with either empty vector and pNL4-3 or a vector expressing gB and pNL4-3 followed by immunoprecipitation of HIV-1 and gB proteins from the culture medium and pellet after ultracentrifugation