Reactivation of latent HIV-1 by new semi-synthetic ingenol esters

Abstract

The ability of HIV to establish long-lived latent infection is mainly due to transcriptional silencing of viral genome in resting memory T lymphocytes. Here, we show that new semi-synthetic ingenol esters reactivate latent HIV reservoirs. Amongst the tested compounds, 3-caproyl-ingenol (ING B) was more potent in reactivating latent HIV than known activators such as SAHA, ingenol 3,20-dibenzoate, TNF-α, PMA and HMBA. ING B activated PKC isoforms followed by NF-κB nuclear translocation. As virus reactivation is dependent on intact NF-κB binding sites in the LTR promoter region ING B, we have shown that. ING B was able to reactivate virus transcription in primary HIV-infected resting cells up to 12 fold and up to 25 fold in combination with SAHA. Additionally, ING B promoted up-regulation of P-TEFb subunits CDK9/Cyclin T1. The role of ING B on promoting both transcription initiation and elongation makes this compound a strong candidate for an anti-HIV latency drug combined with suppressive HAART.

Keywords: HIV; Ingenol; Latency; NF-kB; P-TEFb; PKC; Resting cells.

Fig. 1

Ingenol derivate promotes HIV transcription and virus production. J-Lat cells 6.3 and 8.4 were used as a model of HIV latency. (A) Schematic representation of the novel ingenol ester derivates from Euphorbia tirucalli. (B) and (C) ING B activates HIV transcription in J-Lat cells, clones 6.3 and 8.4, respectively. Cells were exposed to increasing concentrations of ING B and virus activation was evaluated by GFP expression using flow cytometer 24 h after treatment (empty bars). Cytotoxicity was measured using Cell Titer Blue reagent five days post ING B treatment (black lines) and expressed as viability (%). TNF-α (20 ng/ml) was used as positive control (black bars). Error bars represent standard deviation of three independent experiments. (D) ING B treatment promotes HIV production. HIV viral particles were evaluated in the supernatants of J-Lat cells (clones 6.3 and 8.4) exposed to ING B for 48 h by ELISA (HIV-1 p24 Antigen). TNF-α (20 ng/ml) was used as positive control. Mock stands for supernatant of non-treated cells. Error bars represent standard deviation of three experiments.

Fig. 2

HIV reactivation addressed by ING B is mediated by PKC activation. J-Lat cells were pre-treated with different PKC inhibitors at 1 μM for 24 h (Gö 6976, Gö 6983 and Ro-31-8220) before ING B addition (0.32 μM). GFP expression was evaluated 24 h post ING B treatment by flow cytometer. PMA (1 μM) was used as positive control of PKC dependent HIV reactivation. (A) HIV-GFP expression in J-Lat 6.3 cells in the presence of PKC inhibitors. (B) HIV-GFP expression in J-Lat 8.4 cells in the presence of PKC inhibitors. (C) Analysis of cellular localization of HeLa cells expressing different GFP-tagged PKC isoforms by confocal microscopy. HeLa cells were transfected with expressing vectors coding PKC-α (left columns), PKC-δ (central columns) and PKC-γ (right columns) and treated with PMA (1 μM) or ING B (0.32 μM) for 10 min. Untreated cells are labeled as Mock. Cell nuclei are labeled with DAPI (blue color) and PKC-GFP are labeled in green. White arrows are highlighting the sub-membrane location when PKC isoforms are activated. (D) PKC sub-membrane localization was quantified in 200 GFP positive cells for each treatment (Mock non-treated, PMA and ING B). PKC-α, PKC-δ and PKC-γ are represented by black, dark gray and gray bars, respectively. Standard deviation is shown at the top of the bars.

Fig. 3

HIV transcriptional activation by ING B is dependent on NF-κB activation. (A) ING B promotes NF-kB translocation to the nucleus. HeLa cells were treated with ING B (1 μM) for different time points. After that, the cells were fixed and submitted to immunofluorescence using anti-NF-κB antibodies (red color) and DAPI staining (blue color). Cells treated with ING B are presented in lower panel and untreated cells (mock) in upper panel. White arrows highlight NF-κB nuclear translocation. (B) Quantification of NF-κB nuclear translocation in HeLa cells, expressed as fold change (FC) at different time points: 1, 6 and 24 h after ING B treatment (1 μM). PMA (1 μM) was used as positive control (24 h treatment). The fold change was calculated using the average of internalized NF-κB cells of each treatment in comparison to the untreated cells (Mock). 800 HeLa cells were scored and these are results of three independent experiments. (C) Transcription activation by ING B is dependent on intact NF-kB binding site. Jurkat cells were transfected with pBlue3′LTR-Luc (NF-κB WT) and with the construct pBlue3′LTR NF-κB MUT-Luc (NF-κB BS⁻) harboring mutations at NF-κB binding sites that prevent its interaction. After transfections, cells were treated with ING B (0.32 μM). Luciferase assays were performed 24 h post-treatment.

Fig. 4

Non-B subtype LTR promoters are also activated by ING B. (A) Schematic representation of B and C subtype LTR enhancers used. The numbers of NF-κB binding sites are highlighted in the figure. The constructs were denominated Null (without NF-κB binding site), B-LTR (with two canonical NF-κB binding sites), C-LTR and HHC (both with three NF-κB binding sites) and FHHC-LTR (with four NF-κB binding sites). All of these LTR regions described were cloned in the same backbone vector expressing gaussia luciferase. (B) ING B activates luciferase expression controlled by LTR regions from subtype B and C of HIV-1. Luciferase activity in fold change of Jurkat cells transfected with the plasmids cited above and treated with three different activator compounds for 24 h: PMA (1 μM), TNF-α (20 ng/ml) and ING B (0.32 μM). NT stands for non-treated control cells and is represented by white bars.

Fig. 5

ING B compares favorably to other known compounds in HIV reactivation potential. J-Lat cells were treated with different transcriptional activators (ING B, PMA, prostratin, SAHA and ingenol 3,20-dibenzoate) for 24 h. First, J-Lat 6.3 cells (A) and J-Lat 8.4 cells (B) were incubated with each compounds (PMA, prostratin, SAHA and ingenol 3,20-dibenzoate) at 0.32 μM, which is the best concentration of ING B described and percentage of GFP positive cells was determined by FACS. Next, J-Lat 6.3 cells (C) and J-Lat 8.4 cells (D) were incubated with optimal concentrations of each compound (PMA, 20 nM; prostratin, 10 μM; SAHA, 10 μM and ING B, 0.32 μM) and percentage of GFP positive cells were also determined by FACS. Error bars represent the standard deviation of triplicate experiments.

Fig. 6

ING B reactivates latent HIV-1 in primary CD4+ T cells. Naïve CD4+ T cells were isolated from three different healthy donors. CD4+ T cells were activated and infected with an envelope defective HIV-1 virus and returned to a resting state by gradually reducing IL-2 concentration (30–2 U/ml) in the medium. Luciferase activity and levels of activator markers CD38, CD69 and HLA-DR were measured before and at intermediate time points after infection. After resting, cells were stimulated with SAHA, ING B or both. Luciferase activity was measured 24 and 48 h later. (A) Chronological representation of the entire experiment. (B) Average levels of CD38, CD69, HLA-DR and luciferase activity at different time points are indicated. Error bars indicate the standard error of triplicate experiments. Black arrow below the graph indicates the point that HIV infection was performed. (C) Reactivation levels of latent HIV-1 in the resting CD4+ T cells. ING B (1 μM) and SAHA (1 μM) were used alone or in combination as indicated. Luciferase activity was analyzed 24 and 48 h after activation (white and black bars respectively) and is shown as fold change relative to non-treated cells (FC). Error bars indicate the standard deviation of triplicate experiments.

Fig. 7

ING B induces higher levels of P-TEFb components in human primary cells. Naïve T CD4+ cells were isolated from three different healthy donors and treated with ING B (1 μM) for 24 or 48 h. After these periods, P-TEFb components (Cyc T1 and CDK9) expression was analyzed. (A) Cyc T1 (top panel) and CDK9 (middle panel) proteins levels were evaluated by immunoblotting of cell lysates from non-treated (mock) and ING B treated cells for 24 and 48 h. Tubulin (bottom panel) was used as loading control. Non-treated cells are presented in the lines 1, 4 and 7. (B) Quantification of the immunoblots from the three donors is shown as fold change (treated/non-treated cells ratio).