Trans-inhibition of HIV-1 by HIV-lhNef. (A) HEK239T cells were co-transfected with 150 ng of the HIV-1 molecular clone LAI, HIV-lhNef, HIV-CMV, HIV-asCMV or R1 and 150 ng pBluescript (Promega) (upper panel). In the lower panel 150 ng HIV was co-transfected with 150 ng pSuper-shNef, HIV-lhNef, HIV-CMV, HIV-asCMV or R1. 1 ng pRL plasmid, expressing Renilla luciferase from the CMV promoter was added as an internal control for cell viability and transfection efficiency. Transfections were performed with lipofectamine-2000 and 1.5 × 105 cells. Virus production was measured in the culture supernatant 2 days after transfection by CA-p24 ELISA and Renilla expression was measured with the Renilla luciferase assay system (Promega). We plotted the relative percentage of CA-p24/RL, with the HIV + pBluescript transfection set at 100%. Error bars represent the standard deviation from quadruple transfections in three independent experiments. (B) Titration of the HIV-lhNef inhibitor. 500 ng HIV-1 was co-transfected with increasing amounts (0-75-125-250-500 ng) of HIV-lhNef. The total DNA concentration was kept constant by adding pBluescript. Virus production was measured in the culture supernatant 2 days after transfection. Error bars represent the standard deviation in three replicates. This is a representative figure from three transfection experiments with similar results.