Reactivation of HIV-1 from Latency by an Ingenol Derivative from Euphorbia Kansui

Abstract

Cells harboring latent HIV-1 pose a major obstacle to eradication of the virus. The 'shock and kill' strategy has been broadly explored to purge the latent reservoir; however, none of the current latency-reversing agents (LRAs) can safely and effectively activate the latent virus in patients. In this study, we report an ingenol derivative called EK-16A, isolated from the traditional Chinese medicinal herb Euphorbia kansui, which displays great potential in reactivating latent HIV-1. A comparison of the doses used to measure the potency indicated EK-16A to be 200-fold more potent than prostratin in reactivating HIV-1 from latently infected cell lines. EK-16A also outperformed prostratin in ex vivo studies on cells from HIV-1-infected individuals, while maintaining minimal cytotoxicity effects on cell viability and T cell activation. Furthermore, EK-16A exhibited synergy with other LRAs in reactivating latent HIV-1. Mechanistic studies indicated EK-16A to be a PKCγ activator, which promoted both HIV-1 transcription initiation by NF-κB and elongation by P-TEFb signal pathways. Further investigations aimed to add this compound to the therapeutic arsenal for HIV-1 eradication are in the pipeline.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Reactivation of latent HIV-1 by compounds extracted from Euphorbia kansui. (a) C11 cells were treated with various compounds extracted from Euphorbia kansui at three concentrations (0.1 μg/ml, 1 μg/ml and 10 μg/ml). At 48 h post-treatment, the percentage of GFP-positive cells was measured by flow cytometry, which represented the level of HIV-1 transcription. Data show the highest GFP induction of each compound at the three concentrations tested. (b) C11 cells were treated with the 8 selected compounds at the indicated concentrations for 48 h and the percentage of GFP-positive cells was measured. Data show the means ± standard deviations (s.d.) of three independent experiments.

Figure 2

Reactivation of latent HIV-1 in latently infected cells by EK-16A and prostratin. (a) The structure of EK-16A and prostratin. (b) C11 cells and (c) J-Lat 10.6 cells were treated with EK-16A or prostratin at the indicated concentrations. At 48 h post-treatment, the percentage of GFP-positive cells was measured by flow cytometry and dose-dependent curves were shown. Data show the means ± s.d. of three independent experiments.

Figure 3

Effects of EK-16A on cell viability. C11 cells (a) and J-Lat 10.6 cells (b) were treated with EK-16A or prostratin at the indicated concentration for 72 h and then cell viability was measured by CCK-8 kit (Dojindo). The division of OD450 between treated and control groups indicate the percentage of cell viability. Data show the means ± s.d. in three independent experiments.

Figure 4

EK-16A synergizes with other LRAs in reactivating latent HIV-1. J-Lat 10.6 (a) and J-Lat 6.3 cells (b) were treated with EK-16A at the indicated concentrations alone or in combination with prostratin (0.5 μM), 5-Aza (1 μM), JQ1 (0.5 μM), I-Bet151 (0.5 μM), romidepsin (5 nM), or vorinostat (0.5 μM) for 24 h and and the percentage of GFP-positive cells was measured. (c) The Bliss independence model was utilized for calculation of synergy for LRA combinations (See materials and methods). Dotted horizontal line signifies pure additive effect (Δfaxy = 0). Synergy is defined as Δfaxy > 0 while Δfaxy < 0 indicates antagonism. Data show the means ± s.d. of three independent experiments.

Figure 5

EK-16A reactivates HIV-1 from infected resting CD4+ T cells ex vivo. Resting CD4 T cells isolated from five cART-suppressed HIV-1-infected patients were treated with either EK-16A (0.05 μM) or prostratin (1 μM) for 18 h. Intracellular HIV-1 mRNA levels were detected by qRT-PCR and presented as fold induction relative to mock-treated control. **p < 0.01, two-tailed unpaired Student t test, n = 5.

Figure 6

The effects of EK-16A on T cell activation. (a) The effect of EK-16A and prostratin on the expression of CD25 and CD69. Human CD4+ T cells were treated with either EK-16A (0.05 μM) or prostratin (1 μM) for 24 h and the expression of CD25 and CD69 was detected by flow cytometry using antibodies against CD25 and CD69. The percentage of CD69-positive cells (b) and CD25-positive cells (c) was calculated. Data show the means ± s.d. of three independent experiments. (d) Human CD4+ T cells were treated with EK-16A (0.05 μM) or prostratin (1 μM) for 24 h and cell culture supernatant was assayed for cytokine concentrations (pg/ml) by ELISA. Data are representative of three independent experiments.

Figure 7

EK-16A activates HIV-1 mainly through PKCγ. (a) C11 cells were treated with 0.05 μM EK-16A alone (−), or pretreated with Gö 6983 (1 μM), Gö 6976 (1 μM), GF 109203X (1 μM), PKCθ/δ inhibitor (10 μM) or Rottlerin (1 μM) for 3 h, then treated with EK-16A for 48 h before percentage of GFP-positive cells was assessed by flow cytometry. (b) TZM-bl cells were untransfected (−) or transfected with negative control shRNA plasmid (sh-NC) or shRNA plasmids targeting different sequences of PKCγ for 24 h, then treated with 0.05 μM EK-16A for 48 h before firefly luciferase activity was measured. (c) C11 cells were nucleofected with indicated shRNA plasmids for 24 h, then treated with 0.05 μM EK-16A for 72 h before percentage of GFP-positive cells in the shRNA-expressing cells (RFP-positive) was measured by flow cytometry; (d) shows the corresponding data at the different indicated time points. All data represent the mean ± s.d. of three independent experiments.

Figure 8

EK-16A activates HIV-1 by up-regulation of NF-κB and P-TEFb. C11 cells were stimulated with prostratin (1 μM) or ΕΚ-16Α (0.05 μM) for 5, 10 or 30 min, and (a) total cell lysates were probed for the expression of total IκBα or phosphorylated IκBα (p-IκBα) in immunoblots. α-Tubulin expression was used as a control for protein loading. (b) Nuclear extracts were analyzed using antibodies against p65. TATA-binding protein (TBP) expression was used as a control for protein loading. (c) C11 cells were mock-treated or stimulated with prostratin (1 μM) or ΕΚ-16Α (0.05 μM) for 30 min. ChIP assays were performed using anti-p65 antibody and PCR primers for the LTR promoter. The percentage of input for each immunoprecipitation was calculated and the fold occupancy of p65 relative to mock-treatment is shown. Each ChIP experiment was repeated three times to confirm reproducibility of results. (d) C11 cells were stimulated with 0.05 μM EK-16A for 0.5, 1 or 2 h, and total cell lysates were probed for the expression of total CDK9, phosphorylated CDK9 (p-CDK9) or Cyclin T1 in immunoblots. α-Tubulin expression was used as a control for protein loading.

Figure 9

Diagram depicting EK-16A-mediated HIV-1 reactivation by both NF-κB and P-TEFb signaling pathways.