Fig. 7

Sucrose density gradient centrifugation purification of virus reveals the gp120 is not incorporated in viral particles in the presence of HSV-1 gD. 293 cells were co-transfected with either empty pcDNA3.1(+) vector and pNL4-3, a vector expressing gD and pNL4-3, or a vector expressing gB and pNL4-3. At 30 h, the cells were starved for methionine/cysteine, radiolabeled and the culture medium harvested at 48 h post-transfection. Following low speed centrifugation, the culture supernatants were layered onto a 20% sucrose cushion and virus pelleted by ultracentrifugation. The pelleted virus resuspended in DMEM without serum and layered on a discontinuous 20–60% sucrose gradient. The virus was subjected to ultracentrifugation for 20 h (76,000 x g, SW55Ti rotor), 12 fractions were collected, and subjected to immunoprecipitation analysis using anti-HIV-1 antibodies to immunoprecipitated HIV-1 Gag and Env) and appropriate monoclonal antibodies to immunoprecipitate HSV-1 gD or gB. The immunoprecipitates were collected on protein-A-Sepharose, washed, and boiled in sample reducing buffer. The proteins were separated on 7.5% SDS-PAGE and visualized using standard radiographic techniques. a Immunoprecipitation of HIV-1 proteins from gradient fractions of cells co-transfected with empty pcDNA3.1(+) vector and pNL4-3. b Analysis of virus infectivity from various fractions in (a). c Immunoprecipitation of HIV-1 proteins from gradient fractions of cells co-transfected with a vector expressing gD and pNL4-3. d Immunoprecipitation of HSV-1 gD from gradient fractions of cells transfected with a vector expressing gD and pNL4-3. e Immunoprecipitation of gD from gradient fractions of cells transfected with a vector expressing gD. f Analysis of virus infectivity from various fractions in (c, d)