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

Figure 5

LTR-driven gene expression is induced in non-GFP-expressing LAI LTR clones stimulated with various treatments in T-cell clones. Jurkat cells stably transfected with the LAI LTR (WT, 3T, and 5T) were serially diluted in order to obtain one cell in 1 mL of media (approximately 1 cell in 10 wells of a 96-well plate. Cell clone populations were propagated from the single cell stage and then were analyzed using flow cytometry for their basal GFP expression. The Jurkat clonal cell 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. (a) Jurkat cell clones containing all three backbones were treated with TNF-α (20 ng/mL). Levels of GFP expression obtained with the untreated stably transfected cell clone (solid turquoise line) compared to the levels of GFP expression obtained with the treated stably transfected Jurkat cell clone (dashed turquoise line), treated WT Jurkat cells (dashed black line), and untreated WT Jurkat cells (solid black line) are shown. The NE and IE LAI WT Jurkat clones could not be induced into expression. The 3T NE clone could not be induced to drive GFP expression, whereas the IE clone was induced. The NE and IE clones containing the 5T LTR could both be induced into driving GFP expression when stimulated with TNF-α. (b) Jurkat stably transfected cell clones containing the WT, 3T, or 5T LAI LTRs were transfected with 300 ng Tat101 using the Ingenio electroporation solution with an Amaxa nucleofector device and were subsequently analyzed for GFP expression using flow cytometry after 24 hours. Within the context of Tat, untreated refers to transfection with the parental pcDNA3.1 plasmid without the Tat gene (in other words, empty vector). The NE and IE WT LTR and the NE 3T and 5T LTR containing clones could not be transactivated following Tat101 transfection. Expressing clones containing the 3T and 5T LTRs were transactivated to drive higher levels of GFP expression. (c) Stably transfected Jurkat cell clones containing the LAI WT, LAI 3T, or LAI 5T LTRs were treated TSA (400 nM) for 24 hours, along with untreated clone controls as well as untreated and treated Jurkat cells. After 24 hours, cells were analyzed for GFP expression using flow cytometry. The NE 3T and 5T LTR cell clones could be induced into expression with TSA treatment, whereas neither the NE or IE WT cell clones could be induced. (d) Stably transfected Jurkat cell clones containing the LAI WT, LAI 3T, or LAI 5T LTRs were treated with TNF-α (20 ng/mL) and TSA (400 nM) for 24 hours, along with wild type Jurkat cells. After 24 hours, cells were analyzed for GFP expression using flow cytometry. TSA with TNF-α treatment resulted in a small increase in the NE 5T LAI LTR-containing cell clone compared to TSA treatment alone. The combination treatment did not lead to increases in 3T LAI LTR-driven GFP expression within the NE 3T LAI LTR cell clone. (a–d) 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), the treated stably transfected cell clone (dashed turquoise line), untreated WT Jurkat cells (solid black line), and treated WT Jurkat cells (dashed black line) are shown.

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