A flavonoid, luteolin, cripples HIV-1 by abrogation of tat function

Abstract

Despite the effectiveness of combination antiretroviral treatment (cART) against HIV-1, evidence indicates that residual infection persists in different cell types. Intensification of cART does not decrease the residual viral load or immune activation. cART restricts the synthesis of infectious virus but does not curtail HIV-1 transcription and translation from either the integrated or unintegrated viral genomes in infected cells. All treated patients with full viral suppression actually have low-level viremia. More than 60% of treated individuals also develop minor HIV-1 -associated neurocognitive deficits (HAND) due to residual virus and immune activation. Thus, new therapeutic agents are needed to curtail HIV-1 transcription and residual virus. In this study, luteolin, a dietary supplement, profoundly reduced HIV-1 infection in reporter cells and primary lymphocytes. HIV-1inhibition by luteolin was independent of viral entry, as shown by the fact that wild-type and VSV-pseudotyped HIV-1 infections were similarly inhibited. Luteolin was unable to inhibit viral reverse transcription. Luteolin had antiviral activity in a latent HIV-1 reactivation model and effectively ablated both clade-B- and -C -Tat-driven LTR transactivation in reporter assays but had no effect on Tat expression and its sub-cellular localization. We conclude that luteolin confers anti-HIV-1 activity at the Tat functional level. Given its biosafety profile and ability to cross the blood-brain barrier, luteolin may serve as a base flavonoid to develop potent anti-HIV-1 derivatives to complement cART.

Figure 1. Inhibition of HIV-1 by flavonoids.

(A) Chemical structures of flavone and its derivative flavonoids. (B) HIV-1 infection in LTR-luciferase TZM-bl reporter cells after 48 h treatment with luteolin (LN), myricetin (MN), or quercetin (QN), using DMSO as vehicle (Veh) or AZT (positive control). Results were plotted as mean ± SEM of duplicate readings normalized with cell control. (C) TZM-bl cell viability was assessed using WST-8 assay (Dojindo) after treatment for 24 and 48 h with flavonoids (10 µM); in parallel, H₂O₂ was used as positive control. Results are shown as percent viability relative to cell control (n = 3). **p<0.01, ***p<0.001.

Figure 2. Luteolin inhibited HIV-1 infection in Jurkat cells.

A. Viability of TZM-bl, Hela, and Jurkat cells after treatment with different concentrations of luteolin (0–40 µM) for 24–48 h as determined by WST8 -assay (n = 3). B–D. Jurkat cells were pretreated for 30 min with 5 µM or 10 µM luteolin (LN5 and LN10, respectively), vehicle (DMSO), or AZT (positive control) followed by HIV-1 infection for 2 h at 37°C. HIV-1 infection was monitored by (C) GFP expression or (D) virus release in supernatants as determined by p24 ELISA on the 5^th day post-infection. GFP quantification was done by counting 10 random low-power fields and plotted as mean ± SEM (n = 2).

Figure 3. Luteolin inhibited HIV-1 infection in TZM-bl reporter cells.

TZM-bl cells were pretreated for 30 min with 0, 5, and 10 µM luteolin, then infected with HIV-1 for 2 h at 37°C. HIV infection was monitored by (A) GFP expression or (B) virus released in supernatants as determined by p24 ELISA on 3^rd day post- infection (n = 3). C, D. TZM-bl cells were pre- or post-treated with luteolin (10 µM) followed by HIV-1 infection. (C) At 72 h post-infection, HIV-1 infection was monitored by GFP expression. (D) Culture supernatants were analyzed for the virus p24 antigen by ELISA. (n = 2).

Figure 4. Luteolin inhibited HIV-1 infection in primary human lymphocytes.

(A) Viability of primary human lymphocytes after treatment with different concentrations of luteolin (0–40 µM) for 24–48 h as determined by MTT assay (n = 2). (B–D) Luteolin inhibited HIV-1 infection in primary lymphocytes. Primary human lymphocytes were cultured in 12–well culture plates for 6 days in PHA (1%) and IL-2 (10 ng/ml), treated either with luteolin (10 µM) or vehicle, then infected with VSV-HIV-1 or wild–type HIV-1. Viral infection was monitored 2, 4, and 6 days post infection. In parallel, DRB (10 µM) was used as a positive control and DMSO as a vehicle control. (B) The reduction in syncytia formation is evident (white arrows) in luteolin and DRB-treated cells. (C, D), p24 levels in supernatants were monitored by ELISA at (C) 2 days after VSV-HIV infection. (** p<0.01). (D) 4 and 6 days after wild–type HIV-1 infection of lymphocytes (*** p<0.001).

Figure 5. Luteolin inhibited HIV-1 independently of viral entry and reverse transcription steps.

A. Effect of luteolin on viral entry. TZM-bl cells (6×10⁵) in six well tissue culture plates were pretreated with luteolin (5 and 10 µM) or vehicle for 1 h, then infected with HIV-1 infection (p24 = 250 ng/ml) for 2 h at 37°C. After infection, cells were briefly treated with 0.2% trypsin-EDTA and washed extensively to remove cell-membrane-bound virus particles. Six h post-infection, cells were trypsinized and lysed. p24 levels were estimated in cell lysates after normalization of protein concentrations (BCA method) and in HIV-1 infected culture supernatant (HIV-sup). The results are presented as the amount of p24 present per mg of proteins in cell lysates. B. TZM-bl cells (6×10⁵) in six–well tissue culture plates were pretreated with luteolin (10 µM) or DMSO for 30 min, then infected with HIV-1 NLENG1 (p24, 250 ng/ml) for 2 h. At 6 h after infection, cells were treated briefly with 0.2% trypsin and washed. Genomic DNA was harvested from HIV-infected cells. 200 ng of total DNA was used as a template for quantification of viral DNA by real-time PCR using Tat primers and normalized to GAPDH signals. In parallel, 500 ng HIV-1 proviral DNA (pHIV) was transfected as a positive control. C. Jurkat cells (7×10⁵) in six–well culture plates were infected with VSV-HIV-1 (p24 = 250 ng/ml) for 2 h at 37°C, washed twice, and followed up for 24 h. Cells were harvested from 0 to 24 h after infection and viral integration was monitored by Alu-LTR-PCR. D–E. Jurkat cells were infected with VSV-HIV-1 and treated with luteolin (10 µM) or DMSO at 4, 8, and 12 h after infection. The levels of viral infection were monitored by the amount of GFP expression in luteolin- and vehicle-treated HIV-1 infected cells (D). Viral integration was analyzed 24 h post-infection by Alu-LTR PCR (E). VSV-HIV-1 was used as positive control; HIV-1 NL4-3 mutant (D64A) defective in viral DNA integration function (ΔINT HIV) was used as negative control (n = 4).

Figure 6. Luteolin inhibited HIV-1 gene expression independently of viral DNA integration.

A. TZM-bl reporter cells in 12 well culture plates were transfected with HIV-1 plasmid DNA vector expressing GFP, then treated with luteolin (10 µM) or DMSO (Veh). In parallel, TZM-bl cells were infected with VSV-HIV NLENG1 or NLR⁺E⁻ for 2 h, then treated with luteolin (10 µM) or DMSO for the duration of follow up. At 48 h post-transfection or infection, cells were lysed and assayed for luciferase activity (n = 2). B–C. Two hours after Magi cells were transfected with pHIV NLENG1 (150 ng), luteolin (10 µM) was added to them. After 6 h, transfection medium was replaced with fresh medium containing luteolin for 12 or 24 h. At 48 h post-transfection, cells were monitored for GFP expression. Representative pictures are shown (B). Cell supernatants were collected to measure p24 levels (Cs) (n = 2). *** p<0.001, ### p<0.005.

Figure 7. Luteolin inhibited reactivation of latent HIV-1 infection.

(A, B) One million latently HIV-infected THP89 cells seeded per well in 12 well culture plates were pretreated overnight with luteolin (5 or 10 µM) or DMSO, then stimulated with TNF-α (10 ng/ml) and monitored by (A) GFP expression and (B) virus production by p24 ELISA. Similarly, 10 µM luteolin treatments were given from 12 h to 72 h post -TNF-α stimulation. Virus production was monitored by p24 ELISA. DRB (10 µM) was used in parallel as a positive control (n = 3). (C) Luteolin inhibited transactivation of integrated HIV- LTR. SVGA-LTR-GFP reporter cells were transduced with VSV-Tat viral particles for 2 h, then treated with 10 µM luteolin or vehicle and, after 24 h, monitored for GFP expression.

Figure 8. Luteolin inhibited clade B and –C Tat–mediated LTR transactivation in TZM-bl reporter cells.

(A) TZM-bl reporter cells were transfected with Tat expression vector (pcDNA-Tat) and treated after 4 h with different concentrations of either luteolin (0–10 µM) or vehicle and monitored for luciferase activity. (B) TZM-bl cells were transfected with pIRES2-EGFP-Tat-HA (400 ng) and, 24 h later, treated with 0-, 5- and 10- µM luteolin. A Tat-specific siRNA cocktail of 3 siRNAs (300 nM) was co-transfected with Tat expression vector as a positive control. 48 h post-transfection, cells were harvested for Western blot using anti-HA and anti-β actin antibody. (C) TZM-bl cells transfected with HIV-1 subtype-B or -C Tat expression vectors (pcDNA-Tat) were treated with luteolin (10 µM) at 4 h after transfection. In parallel, mutant Tat-47 (Δ 47–56 aa) was used as a negative control. LTR luciferase activity was assessed at 48 h after transfection. Protein levels expressed from Tat expression vectors were monitored by Western blot with anti-HA and anti-β-actin antibody. *** p<0.001. (D) Effect of luteolin treatment on subcellular localization of Tat protein in HeLa cells. Immunostaining showing subcellular localization of Tat protein in HeLa cells after treatment with luteolin (10 µM), DMSO as a vehicle control (DMSO), or untreated (-). IgG was used as isotype antibody control (Isotype). Cells were immunostained for Tat (red), B23/nucleophosmin (green), and nuclei (blue), images were captured at 20× with a Nikon fluorescent microscope.

Figure 9. Proposed schematic representation of anti-HIV activity of luteolin.

After HIV-1 DNA integration into host genome, viral genes are expressed under the control of the HIV-1 long terminal repeat (LTR) as a promoter with the help of viral regulatory protein Tat, which binds with TAR RNA element in the 5′ end of LTR. Luteolin may abrogate Tat-mediated LTR transactivation activity by interfering with pTEF-b binding with LTR or abolish Tat binding; it also may prevent NF-κB activation or inhibition of host factors involved in transcription or inhibition of viral mRNA translation.