FIG. 3.
Analysis of viral proteins in cells and virions by radiolabeling and immunoprecipitation. (A) Virion proteins (from cell supernatant) were immunoprecipitated with a mixture of one anti-RT polyclonal serum and two anti-CAp24 monoclonal antibodies. Lanes: 1, mock-transfected cells; 2, WT pNL4 virion proteins; 3, 4, and 5, RT mutant virion proteins with the indicated mutations. The positions of RTp66, CAp24, and the gag precursor (Pr55gag) are indicated. (B) Viral proteins in cells. Lanes 1 to 4 show proteins immunoprecipitated with the anti-RT polyclonal serum. Lane 1, mock-transfected cells; lanes 2 and 3, transfection with plasmids M230A and L234D, respectively; lane 4, WT pNL4-3 transfection. Lanes 5 to 8 correspond to proteins immunoprecipitated with a mixture of two anti-CAp24 monoclonal antibodies. Lane 5, mock-transfected cells; lanes 6 and 7, transfection with plasmids M230A and L234D, respectively; lane 8, WT pNL4-3 transfection. (C) Viral proteins in cells were immunoprecipitated with a mixture of the anti-RT polyclonal serum and two anti-CAp24 monoclonal antibodies following treatment with Palinavir (22), an HIV PR inhibitor, used at 10 μM during radiolabeling of transfected cells to block PR activity. Lanes: 1, mock-transfected cells; 2 and 3, transfection with WT and mutant L234D plasmids, respectively. Data are representive of three independent experiments.