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. 2013 Feb 1;12(3):452-62.
doi: 10.4161/cc.23309. Epub 2012 Feb 1.

BET Bromodomain-Targeting Compounds Reactivate HIV From Latency via a Tat-independent Mechanism

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Free PMC article

BET Bromodomain-Targeting Compounds Reactivate HIV From Latency via a Tat-independent Mechanism

Daniela Boehm et al. Cell Cycle. .
Free PMC article

Abstract

The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency.

Keywords: BRD2; BRD4; HIV; I-BET; I-BET151; JQ1; MS417; P-TEFb; Tat; latency.

Figures

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Figure 1. JQ1 activates latent HIV. HIV clones R7/E-/GFP and NL4–3/E-/GFP-IRES-nef were derived from pR7-GFP and pNLENG1-EGFP by mutating the Env gene by inserting an early stop codon in the NdeI site. Viral stocks were produced and VSV-G-pseudotyped in 293T cells and titered for p24. Jurkat cells were spininfected with 25 ng of p24 per 106 cells, and GFP cells were collected in two rounds of cell sorting 5 and 15 d after infection. The expanded population of GFP cells, composed of uninfected and latently infected cells, were seeded in 96-well plates and treated with the indicated concentration of drugs in duplicates. The percentage of GFP+ cells was detected after 24 h at the MACSQuant VYB FACS analyzer. Data are expressed as the mean percentage of GFP+ cells, subtracting the average percentage of spontaneous GFP-reactivation in the untreated samples. (A) Jurkat cells containing latent R7/E-/GFP virus were treated with JQ1 or (R)-JQ1 in combination with Prostratin (0.5 μM), SAHA (2.5 μM), TNFα (1 ng/μl) or control at the indicated concentrations for 18 h, followed by flow cytometry analysis. Alone or in combination with Prostratin or TNFα, the BET inhibitor JQ1, but not the stereoisomer control (R)-JQ1, reactivated HIV-1 in a dose-dependent manner. Similar results were seen in Jurkat cells containing latent NL4–3/E-/GFP-IRES -nef (B). Results represent average of two independent experiments.
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Figure 2. The JQ1 effect is Tat-independent. Two latent J-Lat cell lines A2 (containing a LTR-Tat-IRES-GFP construct) or A72 (containing a LTR-GFP construct) were treated with JQ1 or (R)-JQ1 in combination with TNFα or control at the indicated concentrations for 18 h, followed by flow cytometry analysis. (A) In A2 cells JQ1, but not the control (R)-JQ1, reactivated HIV-1 in a dose-dependent manner. Similar results were seen in the Tat-deficient A72 Jurkat cell line (B). Data represent average (± SD) of three independent experiments.
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Figure 3. Reactivation of latent HIV with other bromodomain-targeting compounds. J-Lat cell lines A2 (A) and A72 (B) were treated with JQ1 or two other bromodomain-targeting compounds, I-BET151 and MS417, at the indicated concentrations for 18 h and analyzed by flow cytometry. As indicated, in both A2 and A72 cells, stimulation with all three compounds increased GFP expression. Data represent average (± SD) of three independent experiments.
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Figure 4. Effect of bromodomain-targeting compounds in primary T-cell models of HIV latency. (A) Detection of latently infected cells in sorted GFP-negative Bcl-2-transduced cells. The sorted GFP-Bcl-2-transduced resting CD4+ T cells were treated with stimuli for 24–72 h at 37 þC. Cells treated with 2.5 µg/ml αCD3 and 1 µg/ml αCD28 antibodies were used as positive controls. Reactivation of latent HIV-1 was determined by quantifying % GFP+ cells with a MACSQuant flow cytometer (Milteny Biotech GmbH). Results are expressed as percentage of reactivation in response to αCD3 plus αCD28 activation and represent the average of two independent donors. (B) Effect of bromodomain-targeting compounds in latently infected primary nonpolarized T helper cells. Latently infected T cells were generated using healthy, uninfected CD4+ T cells (DONOR 144) that were ex vivo differentiated into nonpolarized T cells and infected with DHIV-GFP, X4 virus as previously described., Reactivation was monitored by analysis of GFP by flow cytometry 96 h after compound addition. Beads coated with αCD3/αCD28 antibodies were used as positive controls. All compounds were also tested in non-infected cells to distinguish between HIV-1 reactivation and compound fluorescence. Similar results were observed in two independent donors.
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Figure 5. JQ1 enhances transcription burst size. (A and B) Depiction of the experimental model with polyclonal LTR-GFP-containing Jurkat cells. (C and D) Cells treated with JQ1 can change the mean expression level of GFP (hypothesis 1), the variability (or coefficient of variation, CV, defined as the standard deviation over the mean; hypothesis 2) or both, compared with the untreated basal expression state. (E) Genome-wide signatures of JQ1 exposure by time-lapse microscopy. Over 2,000 cells were accounted for and imaged for durations of 12–18 h. JQ1 displays a similar abundance range with elevated noise magnitude compared with the untreated cell population. (F) Histogram/bar representation of the quantified shifts in burst size (or # of mRNA per pulse, T/koff) and the average dwell time in the OFF state (of 1/kon) with and without JQ1 treatment. JQ1 increases burst size and 1/kon.
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Figure 6. The JQ1 effect in A72 cells is dependent on P-TEFb and BRD2. (A) A72 cells were infected with virus containing shRNA constructs targeting cyclin T1 or a non-targeting control. Knockdown of cyclin T1 protein levels are shown by immunoblotting with cyclin T1 or the control α-tubulin antibody. At 4 d after infection, cells were treated with JQ1 or DMSO at the indicated concentrations for 18 h and analyzed by flow cytometry. As indicated, knockdown of cyclin T1 resulted in a decrease in GFP expression under basal condition and in JQ1-treated cells. Average (± SD) of three experiments is shown. (B) A72 cells were infected with virus containing shRNA constructs targeting BRD2, BRD4 or a non-targeting control. At 4 d after infection, cells were treated with JQ1 or DMSO at the indicated concentrations for 18 h and examined by flow cytometry. As indicated, knockdown of BRD2 and BRD4 resulted in an increase in GFP expression. JQ1 treatment enhanced this effect. Results represent average (± SD) of three experiments. Knockdown of BRD2 and BRD4 protein levels were confirmed by immunoblotting with BRD2 and BRD4 antibodies or the control α-tubulin.
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Figure 7. Model: BET proteins restrict HIV transcription in the absence of Tat. JQ1 removes the inhibiting function of BRD2 and BRD4 proteins from latent HIV, a process that may allow both factors to turn into activators of HIV transcription in conjunction with P-TEFb. See text for details.

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