Virus production of the HIV-lhNef escape variants and trans-inhibition of HIV-1. (A) Virus production in HEK293T cells. Cells were transfected with 150 ng pBluescript and 150 ng of the indicated constructs. 1 ng pRL plasmid was added as an internal control. 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 RL expression was measured with the Renilla luciferase assay system (Promega). We plotted the relative percentage of CA-p24/RL, with the transfection HIV + pBluescript set at 100%. Error bars represent the standard deviation from quadruple transfections in two independent experiments. (B) Trans-inhibition of HIV-1 production by the AS escape mutants. HEK293T cells were co-transfected with 150 ng HIV-1 and 150 ng pSuper-shNef, HIV-lhNef, the AS escape mutants or R1. CA-p24 production was measured in the culture supernatant at two days post-transfection. Error bars represent the standard deviation from quadruple transfections in two independent experiments.