Interactions between Nef and AIP1 increase the production of HIV-1 from primary macrophages. (A) Only the wild type Nef protein can rescue the production of mutant viruses in macrophages. Macrophages were derived from PBMCs by adherence to plastic in the presence of 5% human serum. 7 days after differentiation, macrophages were transfected with wild type HIV-1ADA and mutant HIV-1ADAΔ Nef proviruses, or co-transfected with HIV-1ADAΔ Nef provirus with the wild type Nef or mutant NefΔ YPL proteins. 5 days after the transfection, supernatants (S) and cell lysates (L) were examined for the presence of viral particles by the p24 capture ELISA. Bar graphs contain: Black bars, HIV-1ADA alone or the mutant HIV-1ADAΔ Nef provirus with Nef; white bars, the mutant HIV-1ADAΔ Nef provirus; striped bars, the mutant HIV-1ADAΔ Nef provirus with the mutant NefΔ YPL protein. Errors bars denote differences between 5 independent experiments performed with the CaPO4 transfection protocol. (B)Expression of wild type and mutant viruses and wild type and mutant Nef proteins were equivalent in cells.Cell lysates from transfected macrophages were obtained concurrently and processed as in Figs. 1, 2, and 4.