Fig. 10

The gD protein from HSV-2 also restricts the release of infectious HIV-1. 293 cells were co-transfected with either the empty pcDNA3.1(+) vector or one expressing gD, HSV-2 gD (gD2), gB, or UL47 and pNL4-3. At 48 h, the culture supernatants were collected. The levels of infectious virus released into the culture supernatants was determined using TZM-bl cell assays and p24 in the culture supernatants determined using p24 antigen capture assays. a The level of infectious virus released into the culture medium from cells co-transfected with pcDNA3.1(+), gD, HSV-2 gD (gD2), gB, or UL47 and pNL4-3. b The levels of p24 protein produced from cells co-transfected with pcDNA3.1(+), gD, HSV-2 gD (gD2), gB, or UL47 and pNL4-3. c The cell lysates from the above restriction assays were analyzed for the presence of gD, HSV-2 gD (gD2), gB, or UL47 using Western blots and appropriate antibodies. d 293 cells were transfected with empty vector pcDNA3.1(+), pcDNA3.1(+) and pNL4-3, or the vector expressing gD2 and pNL4-3. At 30 h post-transfection, cultures were radiolabelled as per Fig. 8. At 48 h, the culture medium was harvested, centrifuged at low speed to remove cellular debris and layered onto a 20% sucrose cushion. The virus containing medium was subjected to ultracentrifugation to pellet the virus and HIV-1 proteins and gD2 immunoprecipitated using appropriate antibodies. The samples were analyzed by SDS-PAGE and standard radiographic techniques. e, f A companion experiment was performed exactly like d except 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 proteins (Gag and Env) and an appropriate monoclonal antibody to immunoprecipitate HSV-1 gD2. 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. The experiments in a–c were performed at least four times and statistical differences with the pcDNA3.1(+)/HIV-1 control evaluated using a two-tailed Student’s t-test, with p < 0.01 (filled triangle) considered significant