Genistein interferes with SDF-1- and HIV-mediated actin dynamics and inhibits HIV infection of resting CD4 T cells
© Guo et al.; licensee BioMed Central Ltd. 2013
Received: 8 March 2013
Accepted: 10 June 2013
Published: 19 June 2013
Binding of HIV to the chemokine coreceptor CXCR4 mediates viral fusion and signal transduction that promotes actin dynamics critical for HIV infection of blood resting CD4 T cells. It has been suggested that this gp120-mediated actin activity resembles the chemotactic actin dynamics mediated by chemokines such as SDF-1. To determine whether inhibiting SDF-1-mediated chemotactic activity can also inhibit HIV infection, we screened several inhibitors known to reduce SDF-1-mediated chemotaxis of T cells.
We found that a tyrosine kinase inhibitor, genistein, inhibited both SDF-1-mediated chemotaxis and HIV infection of resting CD4 T cells. Genistein was also found to interfere with SDF-1- and HIV-mediated actin dynamics in CD4 T cells. This reduction in actin activity correlates with genistein-mediated inhibition of viral DNA accumulation in resting CD4 T cells. In addition, we also tested two other tyrosine kinase inhibitors, sunitinib and AG1478. Sunitinib, but not AG1478, inhibited HIV infection of resting CD4 T cells. We further tested the safety of genistein in 3 Chinese rhesus macaques (Macaca mulatta), and each animal was given a monotherapy of genistein at 10 mg/kg orally for 12 weeks. No adverse drug effects were observed in these animals.
Our results suggest that novel therapeutic strategies can be developed based on targeting cellular proteins involved in HIV-dependent signaling. This approach can interfere with HIV-mediated actin dynamics and inhibit HIV infection.
Binding of HIV to CD4 and the chemokine coreceptor CXCR4 or CCR5 mediates viral fusion and entry [1–8]. This interaction also triggers the activation of signaling molecules [9–11]. In particular, HIV binding to CXCR4 activates actin regulators such as LIMK1 and cofilin, promoting actin dynamics necessary for viral infection of resting T cells [12–16]. In vivo, chemokine receptor signaling promotes actin dynamics for chemotactic cell migration. Consistently, induction of actin activity by treatment of resting CD4 T cells with chemokines such as CCL2 augments gp120-induced F-actin polymerization and enhances viral DNA synthesis . Similar treatment of memory CD4 T cells with CCL19 triggers cofilin activation and changes in actin filaments, which greatly promote viral nuclear localization and DNA integration [18–20]. In addition, spinoculation of CD4 T cells triggers both cofilin activation and actin dynamics, leading to a great enhancement of HIV DNA synthesis and nuclear migration .
Mechanistically, HIV-mediated actin dynamics are involved in viral early steps, such as entry, post-entry DNA synthesis, and nuclear migration. During viral entry, HIV-1 binding to resting CD4 T cells triggers a rapid and transient actin polymerization through Rac1-PAK1/2-LIMK1-cofilin activation . This rapid actin polymerization transiently blocks CXCR4 internalization to prolong gp120-CXCR4 interaction for membrane fusion to occur . Other actin binding proteins such as Arp2/3, filamin-A, and moesin may also promote actin polymerization and anchor F-actin to membrane proteins to facilitate receptor clustering and viral fusion [22–26]. Following viral entry, the viral reverse transcription complex may be anchored onto F-actin [27–34] for optimal reverse transcription [12, 14, 35]. In addition, HIV-mediated actin treadmilling through CXCR4 signaling and cofilin activity promotes HIV intracellular migration and nuclear localization .
To some extent, HIV-mediated signal transduction through CXCR4 resembles the chemotactic response mediated by chemokines such as SDF-1, the natural ligand for CXCR4 [36, 37]. The major biological response of SDF-1/CXCR4 interaction is to trigger rapid actin activity, which drives directional cell migration. Given that both SDF-1 and HIV require actin dynamics for cell migration and infection, respectively, we speculated that inhibition of certain shared chemotactic pathways between SDF-1 and gp120 may also inhibit HIV infection of resting T cells. Thus, we tested multiple known chemotactic inhibitors of SDF-1 to determine whether these inhibitors can also inhibit HIV. We demonstrate that a tyrosine kinase inhibitor, genistein, known to inhibit SDF-1-mediated chemotaxis, inhibited HIV infection of resting T cells.
Genistein is a tyrosine kinase inhibitor found in a number of plants such as soybeans and flemingia vestita , and is being tested for treatment of cancers such as leukemia [39, 40] and prostate cancer [41, 42]. Genistein has been shown to inhibit human prostate cancer cell migration through inhibiting pro-motility signaling [43, 44]. Genistein similarly inhibits SDF-1-mediated chemotaxis of CD4 T cells , and is suggested to modulate the cellular distribution of actin-binding proteins in human stromal cells by inducing the peri-nuclear accumulation of the actin-binding proteins formin-2 and profilin . In addition, genistein has been shown to inhibit HIV infection of resting CD4 T cells [47, 48] and macrophages by affecting an unknown early step at entry and post-entry . These previous findings have led us to speculate that genistein may inhibit HIV infection of resting CD4 T cells through interference with HIV-mediated actin dynamics. In this report we demonstrate that genistein interferes with HIV-mediated actin dynamics and inhibits viral post-entry DNA synthesis and, to a lesser extent, viral DNA nuclear migration in resting T cells. Our results highlight the possibility that novel therapeutic strategies can be developed to target the HIV-mediated cellular signal transduction to actin dynamics.
Genistein inhibits SDF-1-mediated chemotaxis and HIV infection of resting CD4 T cells
Given that both SDF-1 and HIV trigger rapid actin rearrangement in resting CD4 T cells [12, 50], we asked whether chemotaxis inhibitors can also inhibit gp120-mediated chemotactic signaling and HIV infection of resting T cells. Indeed, the Gαi inhibitor pertussis toxin (PTX) has been shown to inhibit both SDF-1 and gp120-mediated actin dynamics, and HIV-1 infection of resting T cells . Thus, we tested several known SDF-1 inhibitors including the tyrosine kinase inhibitors herbimycin and genistein, and the cyclic nucleotides 8-Br-cAMP and 8-Br-cGMP. These inhibitors have been previously shown to affect SDF-1-mediated memory CD4 T cell movement towards or away from SDF-1 .
We also performed a genistein dosage-dependent assay of SDF-1-mediated chemotaxis, and observed dosage-dependent inhibition in concentrations from 2.5 to 10 μM (Figure 1D). However, at higher concentrations (20 and 40 μM), less inhibition was observed in this donor. Although genistein inhibited chemotaxis at all dosages, the overall relationship between inhibition and drug concentration was not linear. Given that genistein likely targets multiple tyrosine kinases which may antagonize each other, the result was not totally surprising, and may result from differing sensitivities of tyrosine kinases to genistein inhibition.
To determine whether these inhibitors can also inhibit HIV infection of resting CD4 T cells, we pretreated resting CD4 T cells with genistein, herbimycin, 8-Br-cAMP or 8-Br-cGMP, and then infected cells with HIV-1. Following infection for 2 hours, cell-free virus and the inhibitors were washed away, and cells were incubated in the absence of the inhibitors for 5 days, during which productive viral replication does not occur. However, viral replication is inducible upon CD3/CD28 stimulation . As shown in Figure 1E, we activated infected cells with anti-CD3/CD28 beads and observed minimal inhibition of HIV replication by herbimycin, 8-Br-cAMP and 8-Br-cGMP. Nevertheless, we observed a 50% reduction of HIV replication by 3.7 μM genistein (at day 10) in this particular donor. We also performed an experiment on HIV-1 infection at different genistein dosages, and observed dosage-dependent inhibition in concentrations below 5 μM (Figure 1F). However, at higher dosages (10 and 40 μM), the inhibition were less in this donor, similar to the chemotaxis inhibition results in Figure 1D; although genistein inhibited HIV-1 replication at all dosages tested, the overall extent of inhibition was not strictly dosage-dependent. The inhibition of HIV infection did not result from cytotoxicity or inhibition of T cell activation by genistein; when resting CD4 T cells were similarly treated with genistein and activated with CD3/CD28 beads, we did not observe inhibition of T cells activation at all the dosages tested, as judged by the upregulation of the CD25 and CD69 surface receptors (Figure 1G).
Genistein inhibits HIV infection of resting CD4 T cells, viral DNA synthesis, and viral nuclear migration
We further tested the effect of genistein on HIV infection of peripheral blood monocyte-derived macrophages. Cells were pretreated with 37 μM (or 10 μg/ml ) genistein for 1 hour and infected with a primary M-tropic HIV stain, THRO.c/2626 , for 2 hours. Following infection, both genistein and HIV were washed away, and viral replication was monitored. We observed inhibition of HIV by genistein (Figure 2C), similar to a previous report .
We also asked whether other clinical tyrosine kinase inhibitors would be able to inhibit HIV infection of resting T cells, and tested two anti-tumor drugs, sunitinib and AG1478 (tyrphostin). Sunitinib inhibits cellular signaling by targeting multiple receptor tyrosine kinases , whereas AG1478 selectively inhibits epidermal growth factor receptor (EGFR) activation by inhibiting EGFR tyrosine kinase . As shown in Figure 2D, we observed inhibition of HIV-1 infection by sunitinib at 0.2-20 μM in one donor and at 20 μM in a second donor. We observed no inhibition of HIV-1 by AG1478 at all dosages tested (0.2-20 μM) in one donor.
Genistein interferes with SDF-1- and HIV-mediated actin dynamics in resting CD4 T cells
Pre-stimulation of CD4/CXCR4 receptors diminishes genistein-mediated inhibition of HIV infection of resting CD4 T cells
Genistein safety evaluation in rhesus macaques
In this report, we demonstrated that the T cell chemotaxis inhibitor, genistein, interfered with SDF-1-mediated actin dynamics. Similar treatment of resting T cells with genistein also interfered with HIV-1-mediated actin activity and inhibited HIV infection of resting T cells. Genistein is a tyrosine kinase inhibitor found in a number of plants such as soybeans and flemingia vestita , and is being tested for treatment of cancers such as leukemia [39, 40] and prostate cancer [41, 42]. Dietary genistein has also been shown to inhibit metastasis of human prostate cancer in mice [41, 42]. Genistein inhibits human prostate cancer cell motility through inhibiting pro-motility signaling, specifically, by inhibiting the activation of FAK and the p38 MAPK-HSP27 pathway [43, 44]. Genistein has also been suggested to modulate the cellular distribution of actin-binding proteins in human stromal cells by inducing the peri-nuclear accumulation of the actin-binding protein formin-2 and profilin . Although the exact signaling molecules targeted by genistein in HIV infection were not systematically investigated in our study, genistein was found to inhibit the overall phosphorylation of LIMK and cofilin (Figure 5C), two of the main actin regulators involved in T cell mobility and HIV infection [12, 14]. In cells, multiple actin regulatory proteins, such as gelsolin [68, 69], villin , ezrin , cortactin [72–74], Rac1 , and WASP , require tyrosine phosphorylation for activity. Certainly, some of these molecules such as ezrin [23, 24], Rac1 [14, 77], and WASP [25, 26] have also been implicated in HIV infection of CD4 T cells. It is possible that genistein may additionally affect these actin regulators leading to the inhibition of HIV-1 replication in resting CD4 T cells.
Our results on genistein-mediated reduction of both actin activity and HIV infection are consistent with a demonstrated role of early actin dynamics in HIV infection of resting T cells [12, 14, 17, 19, 51]. Our findings are also consistent with previous studies showing that chemokines such as CCL2, CCL19 augment gp120-induced actin dynamics in resting CD4 T cells, which greatly facilitate HIV DNA synthesis and nuclear migration in resting T cells [17, 19]. In addition, our results are aligned with a recent study demonstrating that the N-terminal fragment of the Slit2 protein inhibits X4 and R5 viral infection by binding to the Robo1 receptor and antagonizing the HIV gp120-mediated Rac1-LIMK-cofilin pathway for actin rearrangement . Similar interference of the HIV-mediated actin pathway has also been reported in cannabinoid receptor 2-mediated inhibition of X4 viral infection of primary blood CD4 T cells . Nevertheless, genistein can inhibit multiple cellular kinases, and we did not exclude that the inhibition of HIV replication by genistein is a combined effect with the inhibition of actin signaling.
HIV infection is a chronic disease that requires life-long treatment on a daily basis. Thus, persistent and direct inhibition of actin dynamics through actin inhibitors may ultimately cause severe cytotoxicity. However, drug-mediated interference or diversion of HIV-dependent signal transduction to actin activity is achievable. It is our interest to search for inhibitors that are capable of interfering with virus-mediated actin activity while minimally affecting cellular actin dynamics. It is possible that such inhibitors may not drastically diminish HIV replication in a short-term. However, with long-term treatment and possibly lower viral drug-resistance, persistent depression of viral loads could be achievable. Genistein has the potential to be one of these cellular protein-based anti-HIV drugs with a favorably low cytotoxicity.
In conclusion, in our study, we demonstrated that genistein inhibits HIV infection of CD4 T cells and macrophages. These results may suggest that similar naturally-occurring kinase inhibitors, with little or no-detectable cytotoxicity, may be good candidates for long-term management of HIV infection. Such cellular protein-based therapy may also have additional advantages in combating HIV drug-resistance.
Isolation of resting CD4 T cells and memory CD4 T cells from peripheral blood
Resting CD4 T cells were purified from peripheral blood by two rounds of negative selection as previously described . All protocols involving human subjects were reviewed and approved by the George Mason University IRB. Briefly, for the first round of depletion, we used monoclonal antibodies against human CD14, CD56 and HLA-DR, DP, DQ (BD biosciences). For the second round of depletion, we used monoclonal antibodies against human CD8, CD11b and CD19 (BD biosciences). To purify resting memory CD4 T cells, we used antibodies against human CD8, CD11b, CD19 and CD45RA (BD biosciences) for the second round depletion. Antibody bound cells were depleted by using Dynabeads Pan Mouse IgG (Invitrogen). Purified cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), penicillin (50 U/ml) (Invitrogen), and streptomycin (50 μg/ml) (Invitrogen). Cells were rested overnight before infection or treatment.
Differentiation of macrophages from peripheral blood monocytes
Macrophages were differentiated from human monocytes from the peripheral blood of HIV-1 negative donors as previously described . Briefly, two million peripheral blood mononuclear cells were plated into each well of six-well plates in serum free RPMI medium for one hour. Adherent cells were cultured in RPMI plus 10% heat inactivated fetal bovine serum (FBS) with 10 ng/ml macrophage colony stimulating factor (M-CSF) (R&D System) for two weeks with medium change for every two days.
Virus preparation and infection of T cells and macrophages
Virus stocks of HIV-1NL4-3 and THRO.c/2626  were prepared by transfection of 293T cells with cloned proviral DNA as described . Supernatant was harvested at 48 hours and filtered through a 0.45 μm nitrocellulose membrane. Virus titer (TCID50) was measured by infection of a Rev-dependent indicator cell line, Rev-CEM [82, 83]. For infection of resting CD4 T cells, 103.5 to 104.5 TCID50 units of HIV-1 were used to infect 106 cells. For infection, CD4 T cells were pretreated with genistein, herbimycin, 8-Br-cAMP, or 8-Br-cGMP, incubated with the virus for 2 hours at 37°C, and then washed twice with medium to remove unbound virus and inhibitors. Infected cells were resuspended into fresh RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 μg/ml) at a density of 106 per ml and incubated for 5 days without stimulation. Cells were activated at day 5 with anti-CD3/CD28 magnetic beads at 2 to 4 beads per cell. For the viral replication assay, 10% of infected cells were taken at days 1, 3, 5, 6, 7, 8, and 9 post infection.
For HIV infection of macrophages, cells were pretreated with genistein (10 μg/ml) or DMSO (1%) for 1 hour, infected at 37°C for 2 hours. Infected cells were washed three times and continuously cultured in RPMI plus 10% FBS without M-CSF. Fresh medium was added every two days. Viral replication was monitored by harvesting supernatant. Levels of p24 in the supernatant were measured using Perkin Elmer Alliance p24 antigen ELISA Kit (Perkin Elmer). Plates were kinetically read using an ELx808 automatic microplate reader (Bio-Tek Instruments) at 630 nm.
SIV infection and genistein treatment of rhesus macaques
Three rhesus macaques of Chinese-origin (Macaca mulatta) were used. All animals were housed at the Tulane National Primate Research Center (TNPRC) and maintained in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Research Council. All studies were reviewed and approved by the Tulane Institutional Animal Care and Use Committee (IACUC). All animals were in the chronic phase of SIVmac251 infection with the plasma viral loads in between 102 to 104 copies/ml. Each animal received 10 mg/kg of genistein daily for 12 weeks by oral administration.
Quantification of plasma viral RNA in infected rhesus macaques
Real-Time PCR was performed by the Pathogen Detection and Quantitation Core (PDQC) of Tulane National Primate Research Center. Plasma samples were spiked with armored RNA (aRNA; Asurgen, Austin, TX) and centrifuged at 25,000 x g for 1 hour. Viral RNA (vRNA) was extracted from the pellet with Proteinase K (2.5 mg/ml; Life Tech, Carlsbad, CA) and the High Pure Viral RNA kit (Roche Indianapolis, IN). Eluted vRNA (100 μl) was then subjected to the RNA Clean and Concentrator kit (ZYMO Research, Irvine, CA) and eluted in 50 μl from which 15 μl was reverse transcribed using MultiScribe™ Reverse Transcriptase (Life Tech) in a 50-μL gene specific reaction. Fourteen microliters of cDNA was added to TaqMan gene expression master mix (Life Tech) along with primers and probe targeting the gag region of SIVmac239 and subjected to 40 cycles of qPCR analyses. Fluorescence signals were detected with an Applied Biosystems 7900HT Sequence Detector. Data were captured and analyzed with Sequence Detector Software (Life Tech). Viral copy numbers were calculated by plotting CT values obtained from samples against a standard curve generated with in vitro-transcribed RNA representing known viral copy numbers (and controlled by addition of known copies of aRNA). The limit of detection of the assay is 119 copies per ml of plasma.
Pre-treatment of cells with signaling pathway inhibitors
Unless specified, cells were pre-treated with pertussis toxin (Sigma) (100 ng/ml), genistein (Calbiochem) (1 μg/ml or 3.7 μM) or herbimycin (Sigma) (1 μM) for 1 hour at 37°C, or with 8-Br-cAMP or 8-Br-cGMP (Sigma) (100 μM) for 15 minutes at room temperature, and then infected with HIV.
For chemo-attractant assay, one half million resting CD4 T cells were suspended into 100 μl of RPMI-1640 medium, and then added to the upper chamber of a transwell plate (6.5 mm diameter and 5 μm pore size with a polycarbonate membrane) (Corning). The lower chamber was filled with 600 μl of medium premixed with SDF-1 (R&D systems). The transwell plate was incubated at 37°C for 2 hours, and then the upper chamber was removed and cells in the lower chamber were counted in a Beckman coulter Z2 cell and particle counter.
FITC-Phalloidin staining of F-actin and flow cytometry
One million cells pretreated with genistein (3.7 μM) for 1 hour at 37°C were stimulated with SDF-1 or HIV-1 for various periods of time. Cells were incubated at 37°C in an Eppendorf Thermomixer with gentle agitation (600 rpm) to prevent cells settling at the bottom. F-actin staining using FITC-labeled phalloidin (Sigma) was carried out according to the manufacturer’s recommendation with minor modifications. Briefly, cells were pelleted, fixed and permeabilized with CytoPerm/Cytofix buffer (BD Biosciences) for 20 minutes at room temperature, washed with cold Perm/Wash buffer (BD Biosciences) twice, followed by staining with 5 μl of 0.3 mM FITC-labeled phalloidin (Sigma) for 30 minutes on ice in the dark. After washing twice with cold Perm/Wash buffer, cells were resuspended in 1% paraformaldehyde and analyzed on a FACSCalibur (BD Biosciences).
Nuclear DNA fractionation and real time PCR measurement of HIV DNA
Infected cells were directly lysed in DNA extraction lysis buffer (SV Genomic DNA Isolation System, Promega). Total cellular DNA was extracted and viral total DNA was measured by real time PCR as previously described . The fractionation of viral DNA in memory T cells was conducted as previously described . Briefly, cells were pelleted at 270 x g for 5 minutes in a microfuge at 4°C, washed once with ice-cold PBS, resuspended into ice-cold cell lysis buffer (10 mM Tris-Cl, pH 7.5, 140 mM NaCl, 5 mM KCl, 1% EDTA, 1% NP-40), incubated on ice for 5 to 10 minutes, and then centrifuged at 270 x g for 5 minutes at 4°C to pellet the nuclei. The nuclear pellet was washed once with ice-cold cell lysis buffer and then dissolved in DNA extraction lysis buffer (SV Total RNA Isolation System, Promega). Total cellular DNA was extracted and viral DNA was measured by real time PCR as previously described .
HIV entry assay
The Nef-luciferase-based HIV entry assay was performed as described . Briefly, cells (1 × 106) were infected with 200 ng of Nef-luciferase containing viruses at 37°C for 2 hours, and then washed three times with medium. Cells were resuspended in 0.1 ml of luciferase assay buffer (Promega) and luciferase activity was measured in live cells using a GloMax-Multi Detection System (Promega).
Western blot to detect LIMK and cofilin activation
One million cells were lysed in NuPAGE LDS Sample Buffer (Invitrogen) and separated by SDS-PAGE, and then transferred onto nitrocellulose membranes (Invitrogen). The blots were washed, blocked with Starting Block blocking buffer (Pierce), and incubated overnight with rabbit polyclonal antibodies specific for phospho-LIMK1/2 (1:1000 dilution) or phospho-cofilin (ser3) (1:500 dilution) (Cell Signaling). The blots were washed and then incubated with goat anti-rabbit 800cw labeled antibodies (Li-cor Biosciences) (1:5000 diluted) for 1h at 4°C. The blots were washed three times and scanned with Odyssey Infrared Imager (Li-cor Biosciences). The same blots were also probed with goat anti-GAPDH antibodies (Abcam, ab9483) (1:1000 dilution). The secondary antibody staining was performed using 1:5000 dilution of Rabbit Anti-Goat IgG DyLight 680 antibodies (KPL). The blots were imaged on an ODYSSEY Infrared imager (Li-Cor Biosciences).
Conjugation of antibodies to magnetic beads and stimulation of resting CD4 T cells
Monoclonal antibodies against Human CD3 (clone UCHT1), CD28 (clone CD28.2), CD4 (clone PRA-T4) or CXCR4 (clone 12G5) were from BD Biosciences. Antibodies were conjugated to magnetic beads and used to stimulate resting CD4 T cells as previously described .
Stained cells were imaged using a Zeiss Laser Scanning Microscope, LSM 510 META, with a 40 NA 1.3 or 60 NA 1.4 oil DIC Plan-Neofluar objective. Samples were excited with a laser line, 488 nm for FITC. Images were simultaneously recorded in two channels: channel one, fluorescent emissions from 505 to 530 nm for FITC (green); channel two, DIC. Images were processed and analyzed by the LSM 510 META software.
We thank the George Mason University (GMU) Student Health Center for blood donations; the NIH AIDS Research and Reference Reagent Program, NIAID, NIH for reagents; Ann-Marie Johnson for technical support; the veterinary staff at the Tulane National Primate Research Center; M. Rosen and the donors and riders from the 2010 NYCDC AIDS Ride. This work was supported by NIH Public Health Service Grant grants 1R01AI081568 from NIAID to YW; R01 AI093307-01A1from NIAID to BL; the National Center for Research Resources, and the Office of Research Infrastructure Programs (ORIP) of NIH (through grant OD011104-51). The study was supported in part by the 2010 NYCDC AIDS Ride organized by M. Rosen.
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