Anti-HIV Activities and Mechanism of 12-O-Tricosanoylphorbol-20-acetate, a Novel Phorbol Ester from Ostodes katharinae

Abstract

APOBEC3G is a member of the human cytidine deaminase family that restricts Vif-deficient viruses by being packaged with progeny virions and inducing the G to A mutation during the synthesis of HIV-1 viral DNA when the progeny virus infects new cells. HIV-1 Vif protein resists the activity of A3G by mediating A3G degradation. Phorbol esters are plant-derived organic compounds belonging to the tigliane family of diterpenes and could activate the PKC pathway. In this study, we identified an inhibitor 12-O-tricosanoylphorbol-20-acetate (hop-8), a novel ester of phorbol which was isolated from Ostodes katharinae of the family Euphorbiaceae, that inhibited the replication of wild-type HIV-1 and HIV-2 strains and drug-resistant strains broadly both in C8166 cells and PBMCs with low cytotoxicity and the EC₅₀ values ranged from 0.106 μM to 7.987 μM. One of the main mechanisms of hop-8 is to stimulate A3G expressing in HIV-1 producing cells and upregulate the A3G level in progeny virions, which results in reducing the infectivity of the progeny virus. This novel mechanism of hop-8 inhibition of HIV replication might represents a promising approach for developing new therapeutics for HIV infection.

Keywords: 12-O-tricosanoylphorbol-20-acetate; APOBEC3G; HIV; Vif; antiviral agent; phorbol ester.

Figure 1

Structure of 12-O-tricosanoylphorbol-20-acetate (hop-8) and 12-Deoxyphorbol-13-acetate (prostratin).

Figure 2

Hop-8 inhibited HIV-1 and HIV-2 wild-type, clinical isolates and resistant strains with low cytotoxicity. (A) The cytotoxicity of hop-8 in C8166; (B) The cytotoxicity of hop-8 in peripheral blood mononuclear cells (PBMCs); (C) The antiviral activities of hop-8 against the clinical isolates HIV-1_KM018, and HIV-1_TC-1 were measured in PBMCs; (D) The antiviral activities of hop-8 against wild-type HIV-1_IIIB, HIV-2_CBL-20, and HIV-1_{ΔVif NL4-3} were measured in the C8166 cell line; (E) Antiviral activities of hop-8 against the HIV-1 drug resistant strains HIV-1_A17, HIV-1_{NL4-3 gp41 (36G) V38A, N42T}, HIV-1_RF/V28F/184V, and HIV-1_74V. The levels of p24 in the cell culture supernatant were measured by ELISA. Each data point represents the mean percent inhibition (relative to the positive control) ± standard deviation (bars), n ≥ 2. Data were analyzed with Origin 8.5 (OriginLab, MA, USA).

Figure 3

Hop-8 restored A3G levels in cells undergoing Vif-mediated A3G degradation. (A) Fluorescence photographs were taken with a fluorescent microscope (Leica DMI4000B); (B) Cells were harvested, and EYFP expression in live cells was analyzed with a FACSVerse flow cytometer. Data are expressed as the means ± SD of at least three independent measurements. Statistical comparisons were performed between A3G-EYFP, the Vif positive group (column 2) and other groups with the pair-sample t-test with Origin 8.5. (p < 0.05, significant difference; *: p < 0.05; **: p < 0.01; NS: not significant); (C) pcDNA3.1-APOBEC3G-HA (0.5 μg) was transfected into TRex-hvif-15 cells. The compound (6.75, 1.35, and 0.27 μM) containing 0.1 μg/mL Dox was added 6 h post-transfection. The relative expression levels of A3G-HA and Vif were normalized by the levels of beta-actin. Values are presented as normalized intensities relative to the values of the Dox and A3G-HA positive groups (lane 5). Each data point represents the mean relative quantity ± standard deviation (bars), n ≥ 3.

Figure 4

Hop-8 enhanced the expression of A3G and reduced p24 expression in HIV-1 producing cells and reduced the infectivity of the produced virus. (A) Hop-8 increased the expression of A3G and Vif while reduced the p24 expression in HIV-1 producing cells. The plasmids pcDNA3.1-APOBEC3G-HA (0.125 μg) and pNL4-3 (0.375 μg) were transfected into 2 × 10⁵ 293T cells. The compound was diluted to 6.75, 1.35, and 0.27 μM with DMEM and added to the wells 6 h post-transfection. The relative expression levels of A3G-HA, Vif, p55, and p24 were normalized by the levels of beta-actin. Values are presented as normalized intensities relative to the values of the pNL4-3 and A3G-HA positive groups (lane 8). Each data point represents the mean relative quantity ± standard deviation (bars), n ≥ 3. (B) Hop-8 reduced the infectivity of the progeny virus. The supernatant of NL4-3 was collected and 4 × 10⁴ TZM-bl cells were infected with NL4-3 that contained 20 ng p24. The residual infection was determined using relative luciferase activity. All data represent the means ± standard deviation (bars), n ≥ 3. Data were analyzed by the unpair-sample t-test with Origin 8.5. (p < 0.05, significant difference; *: p < 0.05; **: p < 0.01; NS: not significant). (C) Hop-8 increased the incorporation of A3G in progeny virions. pcDNA-APOBEC3G-HA (1 μg) and pNL4-3 (1 μg) were transfected consecutively or co-transfected into 293T cells in 6-well cell culture plates. Cells were cultured with or without hop-8 (2.7 μM) for 48 h. The supernatant was collected and centrifuged at 2000× g and the cell debris was discarded. The viral particles were lysed with 0.5% Triton-X100. A3G and p24 levels in the supernatant were determined by western blot. The relative expression levels of A3G-HA were normalized by the levels of p24. Each data point represents the mean relative quantity ± standard deviation (bars), n ≥ 3.

Figure 5

Hop-8 upregulated both the protein and mRNA levels of A3G. (A) Hop-8 upregulated A3G protein level regardless of whether Vif was present or absent and had no influence on Vif expression. pcDNA3.1-APOBEC3G-HA and pcDNA3.1-Vif-HA were transfected consecutively or co-transfected into 293T cells and cultured with DMSO, 2.7 μM hop-8, or 10 μM of the proteasome inhibitor MG-132 for 24 h. A3G, Vif, and beta-actin were detected by western blot; (B) A3G mRNA levels of pcDNA3.1-APOBEC3G-HA transfected 293T cells in the presence of the 2.7 μM (dark gray) and 5.4 μM (gray) hop-8, prostratin (2.7 μM) (light gray), or DMSO (black). The cells collected at 4 h post transfection (0 h of compounds treatment) were used as a control. Each data point represents the mean relative quantity (relative to the mock) ± standard deviation (bars), n = 3; (C) A3G mRNA levels in PBMCs which treated with hop-8 (2 μM) (black) or prostratin (2 μM) (gray). Each data point represents the mean relative quantity (relative to the mock) ± standard deviation (bars), n ≥ 3. Data were analyzed by the Mann Whitney test with GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). (p < 0.05, significant difference; *: p <0.05; **: p < 0.01).

Figure 6

Hop-8 did not interfere in the interaction of Vif with A3G, CBF-β, Elongin C, or Cullin 5. (A) Hop-8 did not interfere in Vif and A3G interaction. The plasmid pcDNA3.1-APOBEC3G-HA (3 μg) was transfected into 2 × 10⁶ Trex-hvif-15 cells and treated with 0.1 μg/mL Dox and 2.7 μM hop-8 or DMSO for 48 h. Cells were treated with 10 μM MG-132 for 16 h. Co-IP assays were performed with an anti-HIV1 Vif antibody; (B–D) Hop-8 did not block the interaction between Vif and CBF-β (B), Elongin C (C), or Cullin 5 (D). The plasmids pcDNA3.1-Vif-HA (1.5 μg) and pcDNA3.1-CBF-β-FLAG (1.5 μg) or pcDNA3.1-Elogin C-FLAG or pcDNA3.1-Cullin 5-FLAG were co-transfected into 2 × 10⁶ 293T cells. Cells were treated with 2.7 μM hop-8 or DMSO. Co-IP assays were performed with an anti-HA antibody; (E) The influence of hop-8 on A3G ubiquitination. The plasmids pcDNA3.1-APOBEC3G-HA (1.5 μg) and pUb-MYC (1.5 μg) were co-transfected into 2 × 10⁶ Trex-hvif-15 cells. Cells were treated with 0.1 μg/mL Dox and 2.7 μM hop-8 or DMSO. The Co-IP assay was performed with a rabbit anti-HA antibody.

Figure 7

Schematic of the anti-HIV-1 activity mechanism of hop-8. The process shown with deep green arrows represents a simple schematic of HIV replication. Hop-8 stimulates A3G expression via the PKC pathway. A3G is packaged into the progeny virions and restricts the replication of the progeny virions (shown with purple arrows).