Effect of NNRTIs on alpha complementation and intracellular Gag processing efficiency. (A) 293T cells transfected with a mixture of pCMVω and pCHIV.MAα (lanes 1-2), pCHIV.MAα(PR-) (lanes 3-4), or pCHIV.MAα2PR (lanes 5-6), respectively, were incubated in the presence of DMSO (lanes 1, 3 and 5), or 5 μM EFV (lanes 2, 4 and 6). At 44 h post transfection, cell lysates were harvested and analyzed by immunoblot using antiserum raised against HIV CA (top), as well as for relative β-Gal activity (bottom). CPRG cleavage rates determined as described in materials and methods were normalized to the value obtained for the respective solvent control. (B) 293T cells transfected with a mixture of pCHIV.MAα and pCMVω were grown in the presence of DMSO or 0.25 to 10 μM of the indicated NNRTI, respectively. At 44-48 h post transfection, cell lysates were harvested and analyzed for β-Gal activity. The graph shows mean CPRG cleavage rates and standard deviations from 3-5 transfections each out of three independent experiments. Values were normalized to the cleavage rate obtained for the corresponding solvent control (indicated by a gray line). (C) Lysates of transfected cells grown in the presence of 0.5 μM of the respective inhibitor were analyzed for Gag processing by quantitative immunoblot using antiserum against HIV CA. Data from one representative replicate are shown.