FIG. 4.

Nef expressed from the retroviral vector pHR′CMVNef is sufficient to induce apoptosis in MVECs in a dose-dependent manner. Representative TUNEL staining of MVECs infected with HIV-1-based vectors expressing either Nef or LacZ for 72 h, after which cells were subjected to TUNEL staining, is shown. The amount of virus used varied from 1 to 10 ng of HIV-1 p24 antigen equivalents per ml. TUNEL assays were performed with the in situ cell death detection kit, TMR Red (Roche Diagnostics). The cells were analyzed by fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA). These results are representative of those from experiments performed in triplicate and repeated at least twice.