CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency

Abstract

The establishment of HIV proviral latency requires the creation of repressive chromatin structures that impair the initiation of transcription and restrict RNAP II elongation. We have found that C-promoter binding factor-1 (CBF-1), a CSL (CBF-1, Su(H) and Lag-1)-type transcription factor and key effector of the Notch signaling pathway, is a remarkably potent and specific inhibitor of the HIV-1 LTR promoter. Knockdown of endogenous CBF-1 using specific small hairpin RNAs expressed on lentiviral vectors results in the partial reactivation of latent HIV proviruses, recruitment of RNAP II, loss of histone deacetylases and the concomitant acetylation of histones. An important property of any repressor utilized to establish HIV latency is that it must become displaced or deactivated upon T-cell activation. Consistent with this hypothesis, CBF-1 mRNA and protein levels are highest in quiescent or unstimulated T cells but decline rapidly in response to proliferative stimulation such as activation of the T-cell receptor or treatment with TNF-alpha. We conclude that CBF-1 is a previously overlooked factor that induces transcriptional silencing during the establishment of HIV latency.

Figures

Figure 1

Inhibition of HIV transcription by CBF-1. HeLa (A) and Jurkat T cells (B) carrying latent proviruses expressing the luciferase reporter gene (pHR′-P-Luc; HIV) were co-transfected with between 0 and 3000 ng of a plasmid expressing CBF-1 protein (pcDNA3-CBF-1) along with 200 ng of a Tat-expressing plasmid (pcDNA3-Tat). Control cells were infected with pHR′P-SIN-18-Luc (CMV). In parallel experiments, latently infected HeLa (C) and Jurkat cells (D) were co-transfected with between 0 and 3000 ng of a plasmid expressing NF-κB p50 (pRSV-p50) (West et al, 2001) and 200 ng of pcDNA3-Tat. Latently infected HeLa (E) and Jurkat cells (F) were also co-transfected with between 0 and 3000 ng of a plasmid expressing HDAC-1 and 200ng of pcDNA3-Tat. After 48 h, cell extracts were checked for luciferase activity. Each data set was normalized, with 100% equal to the activity of cells transfected by pcDNA3-Tat alone. As an additional control, basal luciferase levels in the absence of Tat are shown in (A, B).

Figure 2

Complex formation between HIV LTR and CBF-1 protein. Gel-retardation assays were performed using 0–300 ng purified GST-CBF-1 protein and fragments of the HIV LTR. Left: CBF-1 fragment (+76 to +255); right: NF-κB fragment (−160 to −22). LTR fragments with mutated CBF-1-binding sites were used as specificity controls. A fragment of the pcDNA3 vector was used as negative control for CBF-1 binding (far left: control). GST alone was used as control for protein.

Figure 3

CBF-1 blocks HIV transcription in latently infected cells by recruiting HDACs. (A) Lentiviral vectors. (B) Induction of HIV transcription by TNF-α clone E4 cells. The cells were latently infected with the HIV d2EGFP virus (wild-type Tat) and induced for 18 h with 10 ng/ml TNF-α. (C) Induction of HIV transcription by TNF-α in Jurkat clone 2 cells. The cells were latently infected with the HIV mCherry virus (H13L Tat). (D) The distribution of transcription factors on the HIV LTR in clone E4 cells was analyzed by quantitative chromatin immunoprecipitation (ChIP) assays using the indicated antibodies before and after TNF-α stimulation. (E) Control ChIP experiment using the GAPDH gene. Red bars: unstimulated cells; purple bars: +20 ng/ml TNF-α for 24 h.

Figure 4

CBF-1 limits HIV gene expression in newly infected Jurkat T cells. (A) Structure of lentiviral vectors. (B) Western blot showing nuclear levels of CBF-1 in control cells and cells expressing shRNA to CBF-1. Top panel: CBF-1 antibody; bottom panel: Spt-5 antibody loading control. (C) Flow cytometric analysis of Jurkat T-cell lines with lentiviral vectors. Red line: CBF-1 shRNA; purple line: scrambled shRNA control; black line: uninfected cells. After 24 h, the cells were superinfected with HIV mCherry at an m.o.i. of 1.5 and monitored for mCherry expression over the next 72 h.

Figure 5

Knockdown of CBF-1 by shRNA leads to partial induction of latent HIV. (A) Two-color flow cytometric analysis. Jurkat T cells latently infected by HIV mCherry (clone 2 cells) were superinfected by shRNA vectors. Left: clone 2 cells; middle: empty vector control; right: CBF-1 shRNA; top panels: unstimulated cells; bottom panels: cells treated with 20 ng/ml TNF-α for 16 h. (B) Activation of HIV expression following superinfection by vectors expressing scrambled shRNA and CBF-1 shRNA. Black line: uninduced clone 2 cells; blue line: scrambled shRNA; purple line: CBF-1 shRNA; red line: induced by TNF-α. (C) Induction of luciferase expression in Jurkat pHR′P-Luc (HIV) and Jurkat pHR′P-SIN-18-Luc (CMV) by CBF-1 shRNA. The cells were infected with lentiviral vectors carrying shRNA to p50, p65, CBF-1, IKK-β or a scrambled shRNA control.

Figure 6

Knockdown of CBF-1 by shRNA leads to enhanced histone acetylation. The distribution of transcription factors on the HIV LTR in HIV mCherry clone 2 cells superinfected with lentiviral vectors carrying shRNA was analyzed by quantitative chromatin immunoprecipitation (ChIP) assays using the indicated antibodies before and after TNF-α stimulation. Black bars: empty vector (minus shRNA); red bars: CBF-1 shRNA; purple bars: cells treated with 20 ng/ml TNF-α for 24 h.

Figure 7

CBF-1 mRNA and protein are rapidly downregulated in activated T cells. Jurkat cells were activated by treatment with anti-CD3 and anti-CD28 antibodies and the levels of mRNA were accessed by quantitative real-time PCR. (A) CBF-1 mRNA (red) and YY1 (light blue). (B) HIV mRNA (yellow) and β-actin mRNA (dark blue). (C) CBF-1 and Spt5 protein levels were measured by western blotting at various times following T-cell activation.

Figure 8

Model for regulation of HIV latency by CBF-1. In latently infected cells, transcription is blocked by a repressive chromatin structure induced by CBF-1 binding to the HIV LTR. CBF-1 directly recruits histone deacetylases to the promoter, which repress transcription. Subsequently, histone methylases and HP1 proteins establish heterochromatic structures on the HIV LTR. Activation of HIV by stimulation of NF-κB leads to displacement of CBF-1 from the LTR and recruitment of histone acetyltransferases and chromatin-remodeling factors that lead to the formation of an active transcription unit.