Suberoylanilide hydroxamic acid reactivates HIV from latently infected cells

Abstract

Human immunodeficiency virus (HIV) persists in a latent form in infected individuals treated effectively with highly active antiretroviral therapy (HAART). In part, these latent proviruses account for the rebound in viral replication observed after treatment interruption. A major therapeutic challenge is to purge this reservoir. In this study, we demonstrate that suberoylanilide hydroxamic acid (SAHA) reactivates HIV from latency in chronically infected cell lines and primary cells. Indeed, P-TEFb, a critical transcription cofactor for HIV, is released and then recruited to the viral promoter upon stimulation with SAHA. The phosphatidylinositol 3-kinase/Akt pathway is involved in the initiation of these events. Using flow cytometry-based single cell analysis of protein phosphorylation, we demonstrate that SAHA activates this pathway in several subpopulations of T cells, including memory T cells that are the major viral reservoir in peripheral blood. Importantly, SAHA activates HIV replication in peripheral blood mononuclear cells from individuals treated effectively with HAART. Thus SAHA, which is a Food and Drug Administration-approved drug, might be considered to accelerate the decay of the latent reservoir in HAART-treated infected humans.

Figures

FIGURE 1.

SAHA activates HIV replication at the level of transcription. JΔK (A) or TZM-Bl cells (B) were stimulated with increasing concentrations of SAHA for 48 h. A, viral replication was measured in the supernatant of JΔK cells using p24 ELISA. B, cells were lysed, and luciferase activity was measured. All results are presented as fold induction compared with the unstimulated control. C, PBMCs from healthy donors were infected in vitro with HIV-1LAI, and resting CD4+ T cells were isolated after 14 days of culture in the absence of PHA. These isolated cells were cultured in the presence or absence of SAHA (500 nm) for 2 days, and viral replication was assessed using p24 ELISA. D and E, JΔK and TZM-Bl cells were preincubated or not with Akt and PI3K inhibitors, untreated or treated with SAHA (1 μm), and viral replication was assessed using p24 ELISA.

FIGURE 2.

PI3K/Akt pathway is stimulated by SAHA in primary memory T cells. Comparing stimulated over unstimulated cells, fold changes in phosphorylation was analyzed in T cells (CD3+), CD4+ T cells (CD3+CD4+), naive T cells (CD3+CD4+CD45RA+CD27+), memory T cells (CD3+CD4+CD45RA–), and monocytes/macrophages (CD4+CD33+) for PBMCs of healthy donors. Phosphorylation of Akt (A) and ERK1/2 MAPK (B) was analyzed. Data are representative of three independent experiments conducted with cells from three different donors.

FIGURE 3.

SAHA releases active P-TEFb. Jurkat T cells were untreated or treated with SAHA. At 30 min and 1, 2, or 6 h, cell lysates were subjected to glycerol gradient centrifugation. Fractions were collected and proteins precipitated before Western blotting with anti-HEXIM1 (bottom panels) and anti-Cdk9 (top panels) antibodies. Fractions 1–3 contain the active SC, whereas fractions 5–7 contain the inactive LC.

FIGURE 4.

SAHA stimulates the recruitment of RNAPII and P-TEFb to the HIV LTR. A, schematic representation of the HIV provirus integrated into the genome of JΔK cells. B, JΔK cells were preincubated or not for 1 h with the PI3K inhibitor, LY294002 (10 μm), and then stimulated or not with SAHA (1 μm). ChIP analyses were performed using antibodies against RNAPII and CycT1. The recruitment of these transcription factors to the promoter and coding regions was followed by PCR amplification using primer pairs 1 and 2, respectively.

FIGURE 5.

SAHA activates HIV replication in PBMCs from patients treated effectively with HAART. PBMCs from HAART-treated patients with undetectable viremia for more than 1 year were stimulated or not with SAHA at 500 nm for 6 h (A) or with PHA at 3μg/ml (B) for 24 h. Cells were then cultured in the presence of additional stimulated PBMCs, and viral replication was assessed by p24 ELISA over a period of 21 days. Results from 15 patients are presented. Patients were numbered according to their response to stimuli. Cells from patients 1–10 presented activation of HIV replication, whereas cells from patients 11–15 did not present any HIV replication upon the different stimuli used in these studies.