PMC

SUPT16H interferes with association of P-TEFb with Tat-LTR. A, HEK293 cells stably expressing shSUPT16H (sh2) or shNT were transiently transfected with a pQCXIP-FLAG-Tat vector and subjected to IP assays using an anti-FLAG antibody. Endogenous CCNT1 protein level in cell lysate and precipitated samples were analyzed by Western blots using an anti-CCNT1 antibody. B, ChIP assays were performed against CCNT1 in shSUPT16H (sh2) or shNT expressing HEK293 cells that were infected with VSV-G pseudo-typed HIV-1 NL4–3-Luc (dEnv) viruses. The results throughout were from one representative from three independent experiments.

Depletion of FACT proteins enhances reversal of HIV-1 latency in monocytes. A, U1/HIV cells were transduced with pAPM-shSUPT16H (sh2), shSSRP1 (sh1), or shNT. Cell lysate was prepared, separated by SDS-PAGE, and analyzed by Western blots using anti-SUPT16H or anti-SSRP1 antibody. GAPDH protein level was determined using an anti-GAPDH antibody to indicate equal loading of protein samples. The results were from one representative from three independent experiments. B, cDNA samples from the aforementioned cells were subjected to qPCR assays to measure HIV-1 gag mRNA. The level of gag transcripts was normalized to shNT-expressing cells. C and D, cDNA samples from the aforementioned cells were subjected to qPCR assays to measure the HIV-1 initiation and elongation transcripts. The level of viral transcripts was normalized to shNT-expressing cells. E, THP89GFP cells were transduced with pAPM-shSUPT16H (sh2), shSSRP1 (sh1), or shNT. Cells were lysed, separated by SDS-PAGE, and analyzed by Western blots using anti-SUPT16H or anti-SSRP1 antibody. GAPDH protein level was determined using an anti-GAPDH antibody to indicate equal loading of protein samples. The results were one representative from three independent experiments. F and G, the aforementioned cells were treated with DMSO or SAHA (0.5 μm) for 24 h. Cells were analyzed by flow cytometry. GFP-expressing cells were sorted using a defined gate, and a percentage of GFP-positive cells was measured and normalized to shNT-expressing cells. The results throughout are the means of three independent experiments ± S.D. *, p < 0.05 using Student's t test.

FACT proteins associate with Tat-LTR. A, HEK293 cells were transiently transfected with a pQCXIP-FLAG-Tat vector. At 48 h post-transfection, cells were lysed for IP assays using an anti-FLAG or a mIgG antibody. Cell lysate and precipitated protein samples were separated by SDS-PAGE. Protein levels of SUPT16H, SSRP1, or FLAG-Tat were determined by Western blots using their respective antibody. B, IP assays were performed for HEK293 cells that were co-transfected with pCDNA-V5-SSRP1 and pQCXIP-FLAG-Tat vectors. At 48 h post-transfection, cells were lysed and incubated with an anti-V5 or mIgG antibody. Protein level of V5-SSRP1 or FLAG-Tat in cell lysate and precipitated samples was determined using their respective antibody. C and D, HEK293 cells were infected with VSV-G pseudo-typed HIV-1 NL4–3-Luc (dEnv) virus. At 48 h postinfection, cells were cross-linked using formaldehyde, and nuclei were isolated and lysed. Nuclear lysate was sonicated, precleared, and subjected to ChIP assays using anti-SUPT16H (C), anti-SSRP1 (D), or mIgG antibody. Precipitated DNA samples were released, extracted, and analyzed by semiquantitative PCR using primer sets amplifying the HIV-1 LTR promoter or nef region. E, the aforementioned cells were subjected to ChIP assays using anti-CCNT1 or rabbit IgG (rIgG) antibody. F, pCDNA-V5-SSRP1 vector, with pQCXIP-FLAG-Tat or the empty vector, was transfected in HEK293 cells. Co-immunoprecipitation assays were performed for these cells using an anti-V5 antibody. Precipitated protein samples were separated by SDS-PAGE and analyzed for SUPT16H by Western blot. The results throughout were from one representative from three independent experiments.

Depletion of FACT proteins enhances reversal of HIV-1 latency in J-LAT cells. A, pAPM shRNA targeting SUPT16H or SSRP1 were transduced in J-LAT A2 cells. Cells with stable expression of shRNAs were lysed, separated by SDS-PAGE, and analyzed by Western blots using anti-SUPT16H or anti-SSRP1 antibody. The GAPDH protein level was determined using an anti-GAPDH antibody to indicate equal loading of protein samples. The results were one representative from three independent experiments. B and C, aforementioned cells were treated with DMSO, SAHA (0.5 μm), or prostratin (1 μm) for 24 h. The cells were then analyzed by flow cytometry. GFP-expressing cells were sorted using the defined gate, and the percentage of GFP-positive cells was measured and normalized to shNT-expressing cells. *, p < 0.05 using Student's t test. D, cell cycle analysis. J-LAT A2 cells stably expressing SUPT16H or SSRP1 shRNA were stained with propidium iodide and further analyzed by flow cytometry. Cells expressing shNT serve as a negative control. Flow cytometry plots were one representative from three independent experiments. Cell phase percentages were averaged from three independent experiments and represented in a 100% stacked bar graph. SSRP1 depletion led to changes of cell cycle (p < 0.05, Student's t test).

FACT proteins demonstrate similar effect on HTLV-1 transcription. A and B, the vectors HTLV-1 LTR-luciferase, pTK-Renilla, and BC12-Tax, were co-transfected in HEK293 cells stably expressing shRNAs of SUPT16H (A) or SSRP1 (B). At 48 h post-transfection, luciferase activity was measured and normalized to the Renilla signal. The relative light unit (RLU) of shSUPT16H or shSSRP1 expressing cells was normalized to shNT cells. C, HEK293 cells were transfected with pB-His₆-Tax vector. At 48 h post-transfection, cells were lysed and subjected to IP assays using an anti-SUPT16H or mIgG antibody. Cell lysate and precipitated protein samples were separated by SDS-PAGE. Protein level of His₆-Tax was determined by Western blots using a mouse anti-Tax antibody (4C5). The results were one representative from three independent experiments. The results were one representative from three independent experiments. D, HTLV-1 transformed MT-2 cells were stably transduced with pAPM-shSUPT16H (sh2), shSSRP1 (sh1), or shNT. Cells were lysed, separated by SDS-PAGE, and analyzed by Western blots using anti-SUPT16H or anti-SSRP1 antibody. The GAPDH protein level was determined using an anti-GAPDH antibody to indicate equal loading of protein samples. The results were one representative from three independent experiments. E, cDNA samples from the aforementioned cells treated with SAHA (0.5 μm) were subjected to qPCR assays to measure the HTLV-1 gag/pol mRNA. Level of viral transcripts was normalized to shNT cells. The results throughout are the means of three independent experiments ± S.D. *, p < 0.05 using Student's t test.

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. and . 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.

Depletion of FACT proteins increases HIV-1 transcription. A, RIGER method was applied to analyze screens performed using multiple orthologous RNAi reagents. Genes were ranked in order of their RIGER scores, from lowest to highest. RIGER analysis of these screens recognized several known host restriction factors (CCNK, BRD4, and NELFCD), as well as new ones, such as SUPT16H and SSRP1 FACT proteins. B and C, shRNAs (sh1, sh2) targeting SUPT16H or SSRP1 in lentiviral pAPM vector were transduced in HEK293 cells. HEK293 cells stably expressing shRNAs were lysed, separated by SDS-PAGE, and analyzed by Western blots using anti-SUPT16H (B) or anti-SSRP1 (C) antibody. GAPDH protein level was determined using an anti-GAPDH antibody to indicate equal loading of protein samples. The results were one representative from three independent experiments. D and E, HEK293 cells depleted of FACT proteins were infected with VSV-G pseudo-typed HIV-1 NL4–3-Luc (dEnv) viruses. At 24 h postinfection, the cells were subjected to measurement of luciferase activity that was normalized by cell numbers. F and G, vectors of HIV-1 LTR-luciferase, pTK-Renilla, and pCDNA-Tat were co-transfected in HEK293 cells stably expressing shSUPT16H (F) or shSSRP1 (G). At 48 h post-transfection, luciferase activity was measured and normalized to the Renilla signal. The relative light unit (RLU) of shSUPT16H (F) or shSSRP1 (G) expressing cells was normalized to shNT cells. The results throughout are the means of three independent experiments ± S.D. *, p < 0.05 using Student's t test.