Figure 3

Double-variant 3T5T long terminal repeat (LTR) results in altered gene expression in the stably transfected cell lines. U-937 cells were stably transfected with the LAI LTR, cloned within the context of the green fluorescent protein expression vector pEGFP-N1 using the AMAXA Nucleofector System (Lonza, Basel, Switzerland). Mutagenesis was performed to introduce the position 3 C-to-T change at CCAAT/enhancer binding protein (C/EBP) site I (3 T), a position 5 C-to-T change at Sp site III (5 T), or to introduce both the 3 T and 5 T mutations into a single LTR (3T5T). Flow cytometric analysis was used to measure the ability of each LTR to drive GFP expression as compared with their parental (WT) counterparts. Untransfected cells are depicted in gray. The LAI WT LTR is shown in red; LAI 3 T LTR in green; LAI 5 T LTR in orange, and the LAI 3T5T LTR in blue. Under basal conditions, cells expressing the 3T5T LTR expressed an intermediate level of GFP when compared with WT and the other variants that mainly had low GFP expression. Stably transfected cells were treated with TNF-α (20 ng/mL) for 24 hours and then GFP expression was assessed using flow cytometry. The cells expressing the 3T5T LTR resulted in an increase in the high-GFP-expressing population. In addition, cells expressing GFP driven by the 3 T LTR showed a great increase in high-GFP-expressing cells. Stably transfected cells were transfected with Tat86 (300 ng) using the AMAXA Nucleofector System. Tat stimulation of the LTRs was shown to drive a very high level of GFP expression in the 3T5T LTR-GFP-expressing cells, and a small increase in intermediate/high-GFP-expressing cells when driven by the 3 T LTR.