FIGURE 6.

Depletion of FACT proteins enhances reversal of HIV-1 latency in primary T cells. A, procedures are illustrated for generation of primary CD4+ T cell model of HIV-1 latency to study the latency-reversing effect of FACT proteins. It was adapted from Refs. 33 and 34. Colored bars indicate T cell differentiation and activation states. B, primary CD4+ T cells isolated from three donors were cultured ex vivo. Activated CD4+ T cells were transduced with pINDUCER10-shSUPT16H (sh2), shSSRP1 (sh1), or shNT. Cells stably expressing shRNAs were selected by treating cells with puromycin. shRNA expression was induced with doxycycline. Total RNAs were extracted from these cells and analyzed by reverse transcription and qPCR for measuring the transcripts of FACT proteins. Level of SUPT16H or SSRP1 transcript was normalized to shNT-expressing cells for individual donor. C, pINDUCER10-shSUPT16H or shSSRP1 stably transduced primary memory CD4+ T cells were infected with VSV-G pseudo-typed HIV-1 NL4–3-Luc (dEnv) viruses. Cells were kept in long term culture to permit HIV-1 latency. HIV-1 latently infected cells were treated with doxycycline to induce shRNA expression. Luciferase activity was measured for cells depleted of SUPT16H or SSRP1 and normalized to shNT-expressing cells. The results throughout are the means of three independent experiments ± S.D. * indicates p < 0.05 using Student's t test.