Impact of Viral Activators and Epigenetic Regulators on HIV-1 LTRs Containing Naturally Occurring Single Nucleotide Polymorphisms

Figure 1

TNF-α induction of LTR-driven GFP expression is dependent on the basal LTR expression phenotype in hematopoietic progenitor TF-1 cell clones as well as promonocytic U-937 cell clones. Both TF-1 and U-937 cells stably transfected with the LAI LTR (WT, 3T, 5T, and 3T5T) were serially diluted in order to obtain 1 cell in 1 mL of media (approximately one cell in 10 wells of a 96-well plate). Cell clone populations were propagated from the single cell and then were analyzed using flow cytometry for their basal GFP expression. The clonal populations were then designated in one of three categories, nonexpresser (NE), intermediate expresser (IE), and high-expresser (HE), based on their geometric mean fluorescence intensity (MFI) and their percent cell positive values. Each individual cellular clone across all four backbones was treated with TNF-α (20 ng/mL). All experiments were completed in triplicate in three independent experiments. Representative histograms showing levels of GFP expression obtained with the untreated stably transfected cell clone (solid turquoise line) compared to the levels of GFP expression obtained with treated stably transfected cell clone (dashed turquoise line), treated WT TF-1 and U-937 cells (dashed black line), and untreated WT TF-1 and U-937 cells (solid black line) are shown. (a) The NE LAI WT, 3T, and 5T LTR-containing clones could not be induced into driving GFP expression, whereas their GFP-expressing clone counterparts could be induced. All the NE and the IE 3T5T LTR-containing TF-1 clones could be induced to express higher levels of GFP expression. (b) The NE LAI WT, 3T, and 5T LTR-containing clones could not be induced into driving GFP expression, whereas their GFP-expressing clone counterparts could be induced. All of the IE 3T5T LTR-containing clones could be induced to express high levels of GFP.

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