Inhibition of Human Immunodeficiency Virus Type 1 Entry by a Keggin Polyoxometalate

Abstract

Here, we report the anti-human immunodeficiency virus (HIV) potency and underlying mechanisms of a Keggin polyoxometalate (PT-1, K₆HPTi₂W₁₀O₄₀). Our findings showed that PT-1 exhibited highly potent effects against a diverse group of HIV type 1 (HIV-1) strains and displayed low cytotoxicity and genotoxicity. The time-addition assay revealed that PT-1 acted at an early stage of infection, and these findings were supported by the observation that PT-1 had more potency against Env-pseudotyped virus than vesicular stomatitis virus glycoprotein (VSVG) pseudotyped virus. Surface plasmon resonance binding assays and flow cytometry analysis showed that PT-1 blocked the gp120 binding site in the CD4 receptor. Moreover, PT-1 bound directly to gp41 NHR (N36 peptide), thereby interrupting the core bundle formation of gp41. In conclusion, our data suggested that PT-1 may be developed as a new anti-HIV-1 agent through its effects on entry inhibition.

Keywords: CD4; Keggin polyoxometalate; entry inhibition; gp41 NHR; human immunodeficiency virus type 1.

Figure 1

The anti-human immunodeficiency virus (HIV) activity in activated peripheral blood mononuclear cells (PBMCs) and toxicity of the Keggin polyoxometalate PT-1. ConA stimulated PBMCs were incubated with the HIV-1 NL4-3 strain and increasing doses of PT-1(A) or zidovudine (AZT) (B), HIV-1 replication was evaluated by HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) on days 3, 5, 7, and 9 after infection, and the data were expressed as the means ± standard deviations (SD); (C) The cytotoxicity of PT-1 were measured using TZM-bl, MT4, and PBMCs with Cell counting kit -8 (CCK-8) assay; (D) Representative plots of PBMC apoptosis measured by Annexin V-FITC/PI staining and flow cytometry (FACS) analysis. Early apoptotic cells (Annexin-V positive-PI negative) and late apoptotic cells (Annexin-V-PI positive) are shown in the lower right and upper right, respectively; (E) Representative image of mice bone marrow cells smears from vehicle control (a), 200 mg/kg PT-1 (b), 600 mg/kg PT-1 (c), 1800 mg/kg PT-1 (d), and cyclophosphamide positive control group (e). Cells were fixed and stained with Giemsa stain, magnification 1000× polychromatic erythrocytes (PCEs) with one or more nuclei (white arrow) scored. All data represent two independent experiments.

Figure 2

PT-1 inhibited HIV-1 replication at an early stage. (A) Time-of-addition studies were conducted using TZM-bl assay. TZM-bl cells were infected with HIV-1 pREJO4541.67 before PT-1 (1.7 μM), Maraviroc (MVC, 1 μM), Azidothymidine (AZT, 1 μM), T20 (1 μM) and raltegravir (RAL, 1 μM) were added upon HIV-1 inoculation (0 h) or at indicated time points. Luciferase activity was determined 48 h after infection. Comparison of the effects of PT-1 (B) and AZT (C) on the replication of Env and VSV-G pseudovirions. TZM-bl cells were infected with 1000 TCID₅₀ (50% tissue culture infective dose)/mL Env (pREJO4541.67) and VSV-G pseudovirions, respectively. After 48 h of treatment with PT-1 or AZT, inhibition was analyzed by luciferase activity assays. All data were performed in triplicate and repeated in three independent experiments.

Figure 3

PT-1 inhibited gp120/CD4 binding. (A) Surface plasmon resonance (SPR) signals of gp120/CD4 binding. Recombinant gp120 protein was immobilized on a CM5 sensor chip, followed by injection of soluble CD4 (0.11 μM), PT-1 (2 μM), or a pre-incubated mixture of CD4 (0.11 μM) and PT-1 (0.7 μM); (B) SPR sensorgram of PT-1 binding to CD4 binding. CD4 protein was immobilized on a CM5 sensor chip, followed by injection of a serial dilution of PT-1 (0.7–140.81 μM). Gp120 was used as a positive control; (C) FACS profiles of PT-1 binding to the CD4 receptor on MT4 cells. MT4 cells were incubated PT-1 (0.7, 3.5, 17.6 μM) or gp120 (positive control) for 30 min. Cells were then stained with PE-conjugated anti-CD4 RPA-T4 antibodies (D1 domain) for FACS analysis.

Figure 4

PT-1 inhibited HIV gp416-HB formation. (A) SPR sensorgram of PT-1 binding to the gp41 N36 peptide. N36 peptide was immobilized on CM5 sensor chips. PT-1 was injected over the surface at concentrations from 0.7 to 17.6 Nm; (B) N-PAGE analysis of the inhibitory effects of PT-1 on gp41 6-HPB formation by N36 and C34 peptides. Lane 1: C34, lane 2: C34 + N36, lane 3: C34 + N36 + AZT (0.7 mM, negative control), lane 4: C34 + N36 + PT-1 (0.7 μM), lane 5: C34 + N36 + PT-1 (3.5 μM), lane 6: C34 + N36 + PT-1 (17.6 μM). The average relative density of C34+N36 bands of each lane were analyzed (C). All results were obtained from three independent experiments.

Figure 5

Effects of PT-1 on HIV-1 reverse protease, transcriptase and integrase 3′ processing activity. (A) Protease activity was assessed using a commercial kit, and pepstatin A was used as a positive control; (B) Reverse transcriptase activity was analyzed using a commercial kit with colorimetric assays; the percent inhibition was calculated as compared with the reverse transcriptase control. Nevirapine (NVP, 5 μM) was included as a positive control; (C) Integrase 3′-processing activity was analyzed by fluorescence resonance energy transfer. RAL was used as a positive control. All data were means ± standard deviations (SD) and were representative of three independent experiments.