Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcriptional silencing and post-integration latency

Abstract

HIV-1 gene expression is the major determinant regulating the rate of virus replication and, consequently, AIDS progression. Following primary infection, most infected cells produce virus. However, a small population becomes latently infected and constitutes the viral reservoir. This stable viral reservoir seriously challenges the hope of complete viral eradication. Viewed in this context, it is critical to define the molecular mechanisms involved in the establishment of transcriptional latency and the reactivation of viral expression. We show that Suv39H1, HP1gamma and histone H3Lys9 trimethylation play a major role in chromatin-mediated repression of integrated HIV-1 gene expression. Suv39H1, HP1gamma and histone H3Lys9 trimethylation are reversibly associated with HIV-1 in a transcription-dependent manner. Finally, we show in different cellular models, including PBMCs from HIV-1-infected donors, that HIV-1 reactivation could be achieved after HP1gamma RNA interference.

Figure 1

SUV39H1 and HP1γ mediate repression of the HIV-1 LTR in a chromatin-dependent manner. (A) HeLa LTR-Luc and HeLa cells were transfected twice with siRNA as indicated. For HeLa cells, LTR-Luc construct was cotransfected with siRNA in the second round of transfection. Twenty-four hours after the second round of transfection, cells were transduced with GST or GST-Tat as indicated. Expression of Suv39H1 and GAPDH mRNA was analyzed by RT–PCR using specific oligonucleotides (top panel). Twenty-four hours after transduction, extracts were prepared and luciferase activity was measured (lower panels). Results are presented as fold activation relative to GST-treated cells. The mean relative luciferase activities were obtained from three independent experiments. (B) Experiment was performed as in (A), except that siRNA specific for each isoform of HP1 was used. Expression levels of HP1α, β (Supplementary Figure S1) and γ were analyzed by Western blotting using specific antibodies (top panel). Results are presented as fold activation relative to GST-treated cells. The mean relative luciferase activities were obtained from three independent experiments. (C) Total RNA was prepared from cells transfected with control siRNA (Sc) or HP1γ-specific siRNA (experiment shown in (B)). RT–PCR was performed using oligonucleotides specific for the indicated genes. PCR products were analyzed on agarose gels and visualized by ethidium bromide.

Figure 2

Tat-mediated activation of the HIV-1 LTR is associated with reduction of Suv39H1, HP1γ and H3TriMetK9 associated with the LTR and recruitment of coactivators. HeLa LTR-luc cells were treated for 4 hours with GST or GST-Tat. Tat-mediated LTR activation was measured by RT–PCR using luciferase-specific oligonucleotides and GAPDH oligonucleotide as control (A). Chromatin prepared from GST- and GST-Tat-treated cells was subjected to immunoprecipitation with the indicated antibodies (B). As a control, immunoprecipitation was performed in the absence of antibody (Mock). Input and immunoprecipitated DNAs were subjected to real-time PCR using LTR-specific oligonucleotides. The amount of immunoprecipitated material was normalized to the input DNA. Data are representative of three independent experiments. (C) HIV-1 promoters are associated with HP1γ foci in the absence of Tat. U2OS cells containing an integrated HIV-1 reporter were transfected with a Tat expression vector or a control plasmid. The HIV-1 transcription sites were visualized with a fluorescent oligonucleotide probe against the intronic part of the vector (left panels and red color in the merged image), and HP1γ was labelled with a specific antibody (middle panels and green color in the merged image). In the absence of Tat (upper panels), HIV-1 transcription sites colocalized with HP1γ foci with a high frequency (four out of four in the field shown, and 90% of randomly selected cells; n=60). In contrast, in the presence of Tat (lower panels), HIV-1 transcription sites were located in regions containing low concentrations of HP1γ (two out of two in the field shown, and 95% of randomly selected cells; n=60). Note that the contrasts in the RNA images were scaled differently with and without Tat to allow for their simultaneous visualization. Quantification of the signals in the deconvolved images showed that about 20 times more RNA accumulated at the transcription sites in the presence of Tat. Each field was 69 × 54 μm. Insets: zoom of the boxed region in the merged images (4.4 × 4.4 μm).

Figure 3

HP1γ knock-down results in reduction of Suv39H1 and H3TriMetK9 associated with the LTR and recruitment of PCAF and P-TEFb. HeLa LTR-Luc cells were transfected with control Sc siRNA (A, lane 1; B) or HP1γ-specific siRNA (A, lane 2; B). Forty-eight hours post-transfection, cells were harvested. An aliquot of cells was used to analyze luciferase activity (presented as fold activation relative to Sc siRNA-transfected cells) and HP1γ and tubulin expression by Western blotting (A). ChIP assay was performed as in Figure 2 using the indicated antibodies (B). Data are representative of three independent experiments.

Figure 4

Transcriptional activation of the HIV-1 LTR observed after HP1γ knock-down requires Sp1, P-TEFb and PCAF. HeLa LTR-Luc cells were transfected with siRNAs specific for HP1γ, Sp1, CDK9, PCAF, p300 or control (Sc), as indicated. Luciferase activity was measured 48 h after transfection. Expression levels of HP1γ, Sp1, CDK9, PCAF, p300 and HP1α were determined by immunoblotting. Results are presented as fold activation relative to transfection of control siRNA. The mean relative luciferase activities were obtained from three independent experiments.

Figure 5

(A) Knock-down of HP1γ results in increased virus production in U1 cells, a cellular model for HIV-1 latency. U1 cells were transfected with siRNA as indicated in the figure. Efficiency of HP1α, HP1β and HP1γ knock-down was analyzed 48 h after transfection by Western blotting using a specific antibody. Virus production was monitored 48 h post-transfection by measuring p24 antigen in culture supernatant. Cells were treated with TNFα for 12 h when indicated. (B–D) HP1γ-mediated inhibition of HIV-1 replication is established early after virus infection. Jurkat cells were transfected with either control siRNA or HP1γ-, p300- or HP1α-specific siRNA. At 48 h post-transfection, cells were either (B) analyzed for expression levels of HP1γ, HP1α and p300 or (C) infected with HIV-1, and virus replication was monitored every 3 days after infection by measuring p24 viral antigen in culture supernatant or (D) infected with a single-round infectious virus (HIV-1VSV-luc) and virus production was monitored 48 h after infection by measuring luciferase activity in cell extracts.

Figure 6

Knock-down of HP1γ results in faster HIV-1 replication in PBMCs from infected donors. CD4+ T cells purified from HIV-1-infected untreated (A) or HAART-treated (B) patients were transfected with control siRNA or HP1γ-specific siRNA using nucleofection. siRNA-transfected cells were cocultured with activated PBMCs from a healthy donor. Virus replication was monitored every 3 days after co-culture by measuring p24 viral antigen in culture supernatant (P1: patient 1; P2: patient 2). Knock-down of HP1γ was analyzed by Western blotting 48 h post-transfection in cells that were not subjected to coculture.

Figure 7

A proposed model for the involvement of chromatin in the regulation of HIV-1 promoter activity.