Fig 3.

MMQO activates HIV at the transcriptional level independently of genome context. (A) MMQO activates latent HIV in different genomic contexts. Cells of latently infected clones J-Lat E27, A2, and H2 were incubated with increasing concentrations of MMQO for 24 h and analyzed by flow cytometry for GFP-expressing cells and cell viability. (B) MMQO activates a transfected HIV-1 LTR-luciferase reporter. 293T cells were transfected with an HIV-1 LTR-luciferase reporter plasmid. One day after transfection, cells were treated with MMQO (80 μM), EGF (50 ng/ml), PMA (10 nM), TSA (400 nM), or TNF-α (10 ng/ml) for 36 h, followed by cell lysis, protein normalization (5 μg), and luciferase activity assay. Data are expressed as relative light units (RLU). (C) Time course response of HIV-1 transcription to MMQO analyzed by RT-qPCR. J-Lat E27 cells were treated with 80 μM MMQO for the time indicated, and RNA was extracted. PMA (10 nM) and TNF-α (10 ng/ml) treatments for 12 h were added as controls. HIV transcription was measured by RT-qPCR using primers corresponding to the HIV 5′LTR (R-gag) and normalizing with GAPDH. The fold change (FC) between treated and untreated cells is shown. (D) MMQO stimulates HIV transcription initiation. J-Lat E27 cells were untreated or were treated with 80 μM MMQO for 24 h, RNA was extracted, and HIV reactivation was measured by RT-qPCR. Amplicons corresponding to the 5′LTR (R-gag), Tat, GFP, and 3′LTR (U3) were used. GAPDH expression was measured for normalization. To compare between different amplicons, qPCR was performed in parallel from genomic DNA (gDNA). Data are expressed as relative units (RU) (cDNA amplification/GAPDH)/gDNA amplification in a log scale. A scheme of the HIV minigenome with positions of qPCR primers is shown below. Throughout the figure data are represented as means ± SEM (n = 3).