Fig 1. β-catenin modulates HIV transcription in CD4+ T cells via TCF-4 binding at the LTR.

(A) Schematic showing the -143 TCF-4 binding site relative to other transcription factor binding sites on the HIV LTR and with HXB2 numbering, and the LTR luciferase reporter plasmid constructs, containing deleted TCF binding sites shown with X marks at position -143, +186, or both -143 and +186 relative the wildtype HIV BaL transcription start site. (B) CD4+ T cells isolated from n = 5 healthy donors were activated for 24 hrs then nucleofected with LTR reporter constructs. LTR activity was quantified using luciferase relative light units normalized to total protein concentration of the cell lysate. Fold change in LTR activity of the TCF binding site mutants is shown relative to the wildtype HIV BaL LTR control. Nucleofections were performed in duplicate for each donor. (C) CD4+ T cells were nucleofected with LTR reporter constructs as above but were treated with 200 nM adavinint (ADV) 2 hours post-nucleofection. Fold change in LTR activity of wildtype and mutant TCF-4 binding sites are shown relative to cells not treated with ADV. (D) Schematic showing the HXB2 position and sequence of wildtype HIV-REJO TCF binding site compared to the ideal TCF binding site sequence, as well as the mutated TCF binding site in the U3 region of the 5’ LTR of REJO full length molecular clone. (E) CD4+ T cells isolated from n = 6 healthy donors were stimulated for 24 hours then infected with wildtype (WT) or TCF binding site mutant (MUT) HIV REJO via spinoculation with 0.01 MOI virus. Cells were harvested at 24 hours and intracellular HIV transcripts were quantified relative to GAPDH. Fold increase of MUT over WT virus is shown. (F) CD4+ T cells isolated from 4 healthy donors were infected after 24 hours stimulation. 100nM adavivint (ADV) was added following spinoculation, cells were harvested at 24 hours and HIV transcripts quantified as above. Fold change relative of ADV treated cells (red) to DMSO control for wildtype and mutant virus is shown. (G) PBMCs were isolated from 3 healthy donors, stimulated for 3 days, then spinoculated with 0.01 MOI wildtype and TCF binding site mutant virus. Infected cells were harvested at 3, 5, and 7 days post infection, intracellular HIV transcripts were quantified as above. Fold change of TCF binding site mutant to wildtype infected cells harvested on the same day are shown. Columns indicate mean with SEM error bars for all panels. Significance was determined using paired t-tests for all panels, * p<0.05, ** p<0.01.