Figure 2. The leader specific sequences do not act as IRESs. A. Diagram of reporter constructs. 5′ leader DNA sequences from the gag, nef, vif, vpr and vpu mRNAs were inserted between two luciferase genes. Transcription from this DNA construct in cells produces a capped and poly-adenylated dicistronic message. Translation of the upstream cistron (renilla luciferase, RLUC) is cap-dependent while translation of the downstream cistron (firefly luciferase, FLUC) will occur if there is an upstream IRES present. IRES activity is defined as the ratio of FLUC to RLUC light production (relative light units, RLUs). WT constructs contain the entire 5′ leader (common exon plus LSS) while the LSS construct has the common exon sequence removed. B. IRES activity was measured from Jurkat T-cells transfected with dual-luciferase DNA constructs containing WT and LSS sequences (as shown in panel A). IRES activity from each WT leader construct is set at 100%.