Expression of Tat in HeLa P4 cells. A. Top panel depicts the DNA constructs used in the experiments (at least three independent assays were performed). SSL stands for a stable stem-loop to prevent ribosomes translating the Renilla cistron from reading through the Tat coding sequence. The pdualTatcod lacks the 5' UTR of the Tat RNA, except for the 12 nucleotides upstream from the Tat AUG codon (see materials). B. Middle panel shows the activity of newly made Tat, which activates LacZ transcription from the HIV-1 LTR in HeLa P4 cells. This was monitored by the β-galactosidase activity (see methods). C. Lower panel reports the Renilla luciferase activity (5' cistron) for each DNA construct. All values are expressed per μg of total proteins. Integrity of all viral RNA expressed in HeLa P4 cells was assessed by Northern blotting (data not shown).