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 purified human resting CD45RO+/CD45RA- memory CD4 T cells by negative selection (98% pure, measured by flow cytometry, data not shown) , and then similarly stimulated these cells with either SDF-1 or HIV-1NL4-3 for a time course. We measured SDF-1- and HIV-mediated actin dynamics, and observed quick actin polymerization both in SDF-1- and HIV-stimulated memory CD4 T cells, starting at 1 minute post treatment (Figure 1A and B). Next, we treated resting memory CD4 T cells with chemotactic inhibitors, including pertussis toxin (PTX), genistein, herbimycin, 8-Br-cAMP, or 8-Br-cGMP, and tested the inhibition of SDF-1-mediated chemotaxis in a chemotactic trans-well assay (Figure 1C). We observed reduction of memory T cell migration with PTX and genistein, consistent with previous results . However, we did not observe significant inhibition of T cell chemotaxis in the trans-well assay by herbimycin, 8-Br-cAMP, or 8-Br-cGMP in this particular donor.
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
To further confirm that genistein inhibits HIV infection of resting CD4 T cells, we repeated the above experiment (Figure 1E) in another 4 donors and observed inhibition of HIV infection by transient treatment of resting CD4 T cells with genistein during infection (Figure 2A and Additional file 1: Figure S1). Nevertheless, there were clear donor-dependent variations in the degree of inhibition (Figure 2A). We also performed genistein pre-treatment plus one-dose post-infection treatment of resting CD4 T cells, and observed complete inhibition of HIV at all concentrations tested (2.5 to 40 μM) in one donor. In a second donor, we also observed complete inhibition of HIV-1 at concentrations from 10 to 40 μM, and partial inhibition at 2.5 and 5 μM (Figure 2B and Additional file 1: Figure S2).
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.
Previously, Stantchev et al. reported that 5–10 μg/ml (or 18.5-37 μM) genistein inhibited HIV infection of primary human macrophages ; genistein was also found to be non-toxic to cells for these several hours of short treatment at these dosages, and genistein also did not affect the surface expression of CD4 and CCR5 . Interestingly, genistein blocked viral infection of macrophages if added to cells either before, at the time of infection, or immediately after infection, but not 24 hours later, suggesting that genistein-mediated inhibition is at the step of entry and early post-entry . Thus, we also examined the early steps of HIV infection of resting memory CD4 T cells in the presence or absence of genistein. As shown in Figure 3A, we did not observe inhibition of viral entry using a Nef-luciferase based entry assay . We then followed a time course of viral DNA synthesis. HIV reverse transcription in resting CD4 T cells is a biphasic slow process, with an early and a late DNA synthesis phase that peaks at 2–4 hours and 1–2 days respectively [12, 57]. The process of viral DNA synthesis is also accompanied by viral DNA decay in the absence of chemotactic signaling to promote the nuclear entry of newly synthesized viral DNA [12, 19, 58]. As shown in Figure 3B, we observed that viral DNA synthesis peaked at day 1, and then decreased by day 3; in genistein-treated cells, viral DNA synthesis at day 1 was greatly inhibited. We also examined early viral nuclear entry (4 hours) which is promoted by HIV-1 gp120-CXCR4 signaling . We observed a slight early decrease of viral nuclear DNA in genistein-treated cells (Figure 3D). In conclusion, our results suggest that genistein mainly inhibits the slow accumulation of viral DNA in resting CD4 T cells, and, to a lesser extent, viral nuclear migration. Our results are consistent with previous results on HIV infection of macrophages, suggesting that genistein affects early post-entry steps . Although this previous study suggested that genistein may also inhibit viral entry in macrophages , we did not observe inhibition of viral entry in resting memory CD4 T cells using the Nef-luciferease entry assay (Figure 3A). The difference likely resulted from possible different modes of viral entry in these two different primary cell types. It has been shown that HIV can enter macrophages through membrane fusion and a macropinocytosis-like pathway , whereas in blood resting CD4 T cells, the endocytic entry pathway appears to be defective [13, 60]. Genistein may have a different impact on viral entry into these two different cell types.
Genistein interferes with SDF-1- and HIV-mediated actin dynamics in resting CD4 T cells
Given that HIV-mediated actin dynamics play an important role in HIV infection of resting CD4 T cells [12, 14, 19], we speculated that genistein-mediated inhibition of HIV infection may be related to its inhibition of actin activity. The direct effect of genistein on T cell actin dynamics has not been studied although genistein inhibits SDF-1-mediated chemotaxis of memory CD4 T cells (Figure 1A to D) . Genistein has been suggested to inhibit metastasis of cancer cells by inhibiting cell signaling and the redistribution of actin-binding proteins such as formin-2 and profilin . Thus, we measured effects of genistein on SDF-1-mediated actin dynamics in resting memory CD4 T cells which were pre-treated with 3.7 μM genistein for 1 hour. This dosage of genistein slightly increased basal levels of actin density in some donors but not the others (data not shown). Following genistein pre-treatment, cells were treated with SDF-1 for a time course and actin dynamics were measured. As shown in Figure 4A, genistein did not inhibit SDF-1-mediated early actin polymerization (1 min), but it triggered a faster actin depolymerization at later times (after 5 min), which reduced the sustainability of actin polymerization, decreasing the overall actin dynamics. Similar results were observed in another donor (Additional file 1: Figure S3). Confocal microscopy of genistein-treated cells revealed that there was no gross alteration of cell morphology by genistein, but at 60 minutes after SDF-1 treatment, genistein also appeared to increase nuclear actin accumulation in this particular donor (Figure 4B and Additional file 1: Figure S4).
To determine whether genistein similarly affects HIV-mediated actin dynamics, we pre-treated resting memory CD4 T cells with genistein, infected cells with HIV-1, and then measured virus-mediated actin dynamics. As shown in Figure 5A and B, we observed a similar pattern as seen in Figure 4A. Again, genistein did not inhibit HIV-mediated early actin polymerization (0.5-1 minute), but promoted faster actin depolymerization, reducing the overall actin activity (Additional file 1: Figure S5). We also performed Western blots to examine effects of genistein on actin regulators such as the LIM domain kinase (LIMK) and cofilin [12, 14], which mediate actin depolymerization . As shown in Figure 5C, we observed that genistein decreased HIV-1-mediated LIMK and cofilin phosphorylation and activation, disrupting the signaling pathway. Nevertheless, the effect is likely indirect, possibly resulting from inhibition of upstream tyrosine kinases, as LIMK1/2 and cofilin are phosphorylated on threonine 508/505 and serine 3, respectively [62, 63].
Pre-stimulation of CD4/CXCR4 receptors diminishes genistein-mediated inhibition of HIV infection of resting CD4 T cells
Genistein is a general tyrosine kinase inhibitor, and it is likely that genistein indirectly affected SDF-1- and HIV-1-mediated actin dynamics through inhibition of tyrosine kinases that are involved in actin dynamics. Previously, we have demonstrated that pre-stimulation of resting CD4 T cells with anti-CD4/CXCR4 beads triggers cell signaling and actin reorganization that enhances HIV-1 infection of resting T cells . This low-level stimulation (two beads per cell) does not block HIV entry or activate T cells, but greatly promotes actin dynamics that eliminate the inhibitory effect of the actin modulator jasplakinolide (at 120 and 600 nM) on HIV infection of resting T cells . We speculated that similar stimulation of actin dynamics in resting CD4 T cells with anti-CD4/CXCR4 beads may also overcome genistein inhibition of HIV infection. As expected, we observed a complete loss of the inhibition by genistein when cells were pre-stimulated with anti-CD4/CXCR4 beads (Figure 6A to D), whereas in unstimulated cells, genistein inhibited over 99% of viral replication in CD4 T cells of the same donor (at day 8) (Figure 6C and D). Similar results were observed in another donor, although this donor demonstrated less inhibition of HIV-1 by 10 μM genistein (Figure 6E and F). These results are consistent with our hypothesis that genistein inhibits HIV infection through interference of the CD4/CXCR4-mediated receptor signaling that leads to actin dynamics.
Genistein safety evaluation in rhesus macaques
Genistein is naturally made in a number of plants, such as soybeans, and its consumption is associated with a lower incidence of metastatic prostate cancer in southeast Asians who subsist on a soybean-based diet [41, 64–67]. The average steady-state blood levels of genistein in Japanese men, who subsist on a soy-based diet, were 0.28 μM . In a phase I human clinical trial, subjects were given genistein (43-90% pure) at 2–8 mg/kg orally, and no significant cytotoxicities were observed among these cancer patients . The subjects sustained a maximal total plasma genistein concentration between 4.3 to 16.3 μM, with a drug half-life of 15 to 22 hours . Based on this phase I study, we also initiated a preliminary animal trial to evaluate the safety of daily administration of genistein. Three rhesus macaques of Chinese-origin (Macaca mulatta) were chronically infected with SIVmac251 with plasma viral loads between 102 to 104 copies/ml. Each animal was given a monotherapy of genistein at 10 mg/kg orally for 12 weeks. We did not observe adverse effects on any of these animals, and their CD4 T cell counts and percentages remained stable in these 12 weeks (Figure 7A and B). We also performed a one-time measurement of plasma viral load at the end of the 12 weeks. Two of the animals (PO15 and HG40) had a reduction of viral load to undetectable level, whereas the third animal (IV30) had no reduction (Figure 7C). Given the donor variations and dosage-independent inhibition of HIV observed in our in vitro CD4 T cell cultures, IV30 may need longer treatment or a different genistein dosage. However, we do not excluded that drug resistance might also develop, although it is expected to be more difficult for cellular targets. Further studies are certainly needed to address the in vivo efficacy of genistein, and to define optimal dosages for maximal viral inhibition in individual animals.
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.
National Center for Biodefense and Infectious Diseases, Department of Molecular and Microbiology, George Mason University
Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Disease, NIH, Twinbrook Facility
Division of Comparative Pathology, Tulane National Primate Research Center, Tulane University Health Sciences Center
Klatzmann D, Champagne E, Chamaret S, Gruest J, Guetard D, Hercend T, Gluckman JC, Montagnier L: T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV.Nature 1984, 312:767–768.PubMedView Article
Dalgleish AG, Beverley PC, Clapham PR, Crawford DH, Greaves MF, Weiss RA: The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.Nature 1984, 312:763–767.PubMedView Article
Feng Y, Broder CC, Kennedy PE, Berger EA: HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.Science 1996, 272:872–877.PubMedView Article
Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, Berger EA: CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1.Science 1996, 272:1955–1958.PubMedView Article
Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, et al.: The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates.Cell 1996, 85:1135–1148.PubMedView Article
Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton RE, Hill CM, et al.: Identification of a major co-receptor for primary isolates of HIV-1.Nature 1996, 381:661–666.PubMedView Article
Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, Parmentier M, Collman RG, Doms RW: A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.Cell 1996, 85:1149–1158.PubMedView Article
Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Maddon PJ, Koup RA, Moore JP, Paxton WA: HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.Nature 1996, 381:667–673.PubMedView Article
Weissman D, Rabin RL, Arthos J, Rubbert A, Dybul M, Swofford R, Venkatesan S, Farber JM, Fauci AS: Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor.Nature 1997, 389:981–985.PubMedView Article
Davis CB, Dikic I, Unutmaz D, Hill CM, Arthos J, Siani MA, Thompson DA, Schlessinger J, Littman DR: Signal transduction due to HIV-1 envelope interactions with chemokine receptors CXCR4 or CCR5.J Exp Med 1997, 186:1793–1798.PubMedView Article
Wu Y, Yoder A: Chemokine coreceptor signaling in HIV-1 infection and pathogenesis.PLoS Pathog 2009, 5:e1000520.PubMedView Article
Yoder A, Yu D, Dong L, Iyer SR, Xu X, Kelly J, Liu J, Wang W, Vorster PJ, Agulto L, et al.: HIV envelope-CXCR4 signaling activates cofilin to overcome cortical actin restriction in resting CD4 T cells.Cell 2008, 134:782–792.PubMedView Article
Yu D, Wang W, Yoder A, Spear M, Wu Y: The HIV envelope but not VSV glycoprotein is capable of mediating HIV latent infection of resting CD4 T cells.PLoS Pathog 2009, 5:e1000633.PubMedView Article
Vorster PJ, Guo J, Yoder A, Wang W, Zheng Y, Xu X, Yu D, Spear M, Wu Y: LIM kinase 1 modulates cortical actin and CXCR4 cycling and is activated by HIV-1 to initiate viral infection.J Biol Chem 2011, 286:12554–12564.PubMedView Article
Wu Y: The co-receptor signaling model of HIV-1 pathogenesis in peripheral CD4 T cells.Retrovirology 2009, 6:41.PubMedView Article
Wu Y, Yoder A, Yu D, Wang W, Liu J, Barrett T, Wheeler D, Schlauch K: Cofilin activation in peripheral CD4 T cells of HIV-1 infected patients: a pilot study.Retrovirology 2008, 5:95.PubMedView Article
Campbell GR, Spector SA: CCL2 increases X4-tropic HIV-1 entry into resting CD4+ T cells.J Biol Chem 2008, 283:30745–30753.PubMedView Article
Saleh S, Solomon A, Wightman F, Xhilaga M, Cameron PU, Lewin SR: CCR7 ligands CCL19 and CCL21 increase permissiveness of resting memory CD4+ T cells to HIV-1 infection: a novel model of HIV-1 latency.Blood 2007, 110:4161–4164.PubMedView Article
Cameron PU, Saleh S, Sallmann G, Solomon A, Wightman F, Evans VA, Boucher G, Haddad EK, Sekaly RP, Harman AN, et al.: Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton.Proc Natl Acad Sci USA 2010, 107:16934–16939.PubMedView Article
Wu Y: Chemokine control of HIV-1 infection: beyond a binding competition.Retrovirology 2010, 7:86.PubMedView Article
Guo J, Wang W, Yu D, Wu Y: Spinoculation triggers dynamic actin and cofilin activity facilitating HIV-1 infection of transformed and resting CD4 T cells.J Virol 2011, 85:9824–9833.PubMedView Article
Jimenez-Baranda S, Gomez-Mouton C, Rojas A, Martinez-Prats L, Mira E, Ana Lacalle R, Valencia A, Dimitrov DS, Viola A, Delgado R, et al.: Filamin-A regulates actin-dependent clustering of HIV receptors.Nat Cell Biol 2007, 9:838–846.PubMedView Article
Naghavi MH, Valente S, Hatziioannou T, de Los Santos K, Wen Y, Mott C, Gundersen GG, Goff SP: Moesin regulates stable microtubule formation and limits retroviral infection in cultured cells.EMBO J 2007, 26:41–52.PubMedView Article
Barrero-Villar M, Cabrero JR, Gordon-Alonso M, Barroso-Gonzalez J, Alvarez-Losada S, Munoz-Fernandez MA, Sanchez-Madrid F, Valenzuela-Fernandez A: Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes.J Cell Sci 2009, 122:103–113.PubMedView Article
Komano J, Miyauchi K, Matsuda Z, Yamamoto N: Inhibiting the Arp2/3 complex limits infection of both intracellular mature vaccinia virus and primate lentiviruses.Mol Biol Cell 2004, 15:5197–5207.PubMedView Article
Harmon B, Campbell N, Ratner L: Role of Abl kinase and the Wave2 signaling complex in HIV-1 entry at a post-hemifusion step.PLoS Pathog 2010, 6:e1000956.PubMedView Article
Rey O, Canon J, Krogstad P: HIV-1 Gag protein associates with F-actin present in microfilaments.Virology 1996, 220:530–534.PubMedView Article
Liu B, Dai R, Tian CJ, Dawson L, Gorelick R, Yu XF: Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin.J Virol 1999, 73:2901–2908.PubMed
Wilk T, Gowen B, Fuller SD: Actin associates with the nucleocapsid domain of the human immunodeficiency virus Gag polyprotein.J Virol 1999, 73:1931–1940.PubMed
Ibarrondo FJ, Choi R, Geng YZ, Canon J, Rey O, Baldwin GC, Krogstad P: HIV type 1 Gag and nucleocapsid proteins: cytoskeletal localization and effects on cell motility.AIDS Res Hum Retroviruses 2001, 17:1489–1500.PubMedView Article
Hottiger M, Gramatikoff K, Georgiev O, Chaponnier C, Schaffner W, Hubscher U: The large subunit of HIV-1 reverse transcriptase interacts with beta-actin.Nucleic Acids Res 1995, 23:736–741.PubMedView Article
Turlure F, Devroe E, Silver PA, Engelman A: Human cell proteins and human immunodeficiency virus DNA integration.Front Biosci 2004, 9:3187–3208.PubMedView Article
Niederman TM, Hastings WR, Ratner L: Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix.Virology 1993, 197:420–425.PubMedView Article
Fackler OT, Kienzle N, Kremmer E, Boese A, Schramm B, Klimkait T, Kucherer C, Mueller-Lantzsch N: Association of human immunodeficiency virus Nef protein with actin is myristoylation dependent and influences its subcellular localization.Eur J Biochem 1997, 247:843–851.PubMedView Article
Bukrinskaya A, Brichacek B, Mann A, Stevenson M: Establishment of a functional human immunodeficiency virus type 1 (HIV-1) reverse transcription complex involves the cytoskeleton.J Exp Med 1998, 188:2113–2125.PubMedView Article
Balabanian K, Harriague J, Decrion C, Lagane B, Shorte S, Baleux F, Virelizier JL, Arenzana-Seisdedos F, Chakrabarti LA: CXCR4-tropic HIV-1 envelope glycoprotein functions as a viral chemokine in unstimulated primary CD4+ T lymphocytes.J Immunol 2004, 173:7150–7160.PubMed
Guo J, Xu X, Yuan W, Jin T, Wu Y: HIV gp120 is an aberrant chemoattractant for blood resting CD4 T cells.Curr HIV Res 2012, 10:636–642.PubMed
Kaufman PB, Duke JA, Brielmann H, Boik J, Hoyt JE: A comparative survey of leguminous plants as sources of the isoflavones, genistein and daidzein: implications for human nutrition and health.J Altern Complement Med 1997, 3:7–12.PubMedView Article
Raynal NJ, Momparler L, Charbonneau M, Momparler RL: Antileukemic activity of genistein, a major isoflavone present in soy products.J Nat Prod 2008, 71:3–7.PubMedView Article
Raynal NJ, Charbonneau M, Momparler LF, Momparler RL: Synergistic effect of 5-Aza-2'-deoxycytidine and genistein in combination against leukemia.Oncol Res 2008, 17:223–230.PubMedView Article
Lakshman M, Xu L, Ananthanarayanan V, Cooper J, Takimoto CH, Helenowski I, Pelling JC, Bergan RC: Dietary genistein inhibits metastasis of human prostate cancer in mice.Cancer Res 2008, 68:2024–2032.PubMedView Article
Takimoto CH, Glover K, Huang X, Hayes SA, Gallot L, Quinn M, Jovanovic BD, Shapiro A, Hernandez L, Goetz A, et al.: Phase I pharmacokinetic and pharmacodynamic analysis of unconjugated soy isoflavones administered to individuals with cancer.Cancer Epidemiol Biomarkers Prev 2003, 12:1213–1221.PubMed
Xu L, Bergan RC: Genistein inhibits matrix metalloproteinase type 2 activation and prostate cancer cell invasion by blocking the transforming growth factor beta-mediated activation of mitogen-activated protein kinase-activated protein kinase 2–27-kDa heat shock protein pathway.Mol Pharmacol 2006, 70:869–877.PubMedView Article
Huang X, Chen S, Xu L, Liu Y, Deb DK, Platanias LC, Bergan RC: Genistein inhibits p38 map kinase activation, matrix metalloproteinase type 2, and cell invasion in human prostate epithelial cells.Cancer Res 2005, 65:3470–3478.PubMed
Poznansky MC, Olszak IT, Foxall R, Evans RH, Luster AD, Scadden DT: Active movement of T cells away from a chemokine.Nat Med 2000, 6:543–548.PubMedView Article
Shieh DB, Li RY, Liao JM, Chen GD, Liou YM: Effects of genistein on beta-catenin signaling and subcellular distribution of actin-binding proteins in human umbilical CD105-positive stromal cells.J Cell Physiol 2010, 223:423–434.PubMed
Wu Y, Yoder RAC, Kelly J, Yu D: Composition and methods for detecting and treating HIV infections. U.S. Provisional Patent Application; 2006. No. 60/797,745
Wu Y, Yoder RAC, Kelly J, Yu D: Compositions and methods for detecting and treating HIV infections.US Patent 2010, 7:662,547.
Stantchev TS, Markovic I, Telford WG, Clouse KA, Broder CC: The tyrosine kinase inhibitor genistein blocks HIV-1 infection in primary human macrophages.Virus Res 2007, 123:178–189.PubMedView Article
Nishita M, Aizawa H, Mizuno K: Stromal cell-derived factor 1alpha activates LIM kinase 1 and induces cofilin phosphorylation for T-cell chemotaxis.Mol Cell Biol 2002, 22:774–783.PubMedView Article
Wang W, Guo J, Yu D, Vorster PJ, Chen W, Wu Y: A dichotomy in cortical actin and chemotactic actin activity between human memory and naive T cells contributes to their differential susceptibility to HIV-1 infection.J Biol Chem 2012, 287:35455–35469.PubMedView Article
Wu Y, Marsh JW: Selective transcription and modulation of resting T cell activity by preintegrated HIV DNA.Science 2001, 293:1503–1506.PubMedView Article
Keele BF, Giorgi EE, Salazar-Gonzalez JF, Decker JM, Pham KT, Salazar MG, Sun C, Grayson T, Wang S, Li H, et al.: Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection.Proc Natl Acad Sci USA 2008, 105:7552–7557.PubMedView Article
Faivre S, Demetri G, Sargent W, Raymond E: Molecular basis for sunitinib efficacy and future clinical development.Nat Rev Drug Discov 2007, 6:734–745.PubMedView Article
Zhu XF, Liu ZC, Xie BF, Li ZM, Feng GK, Yang D, Zeng YX: EGFR tyrosine kinase inhibitor AG1478 inhibits cell proliferation and arrests cell cycle in nasopharyngeal carcinoma cells.Cancer Lett 2001, 169:27–32.PubMedView Article
Saeed MF, Kolokoltsov AA, Davey RA: Novel, rapid assay for measuring entry of diverse enveloped viruses, including HIV and rabies.J Virol Methods 2006, 135:143–150.PubMedView Article
Zack JA, Arrigo SJ, Weitsman SR, Go AS, Haislip A, Chen IS: HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure.Cell 1990, 61:213–222.PubMedView Article
Zhou Y, Zhang H, Siliciano JD, Siliciano RF: Kinetics of human immunodeficiency virus type 1 decay following entry into resting CD4+ T cells.J Virol 2005, 79:2199–2210.PubMedView Article
Carter GC, Bernstone L, Baskaran D, James W: HIV-1 infects macrophages by exploiting an endocytic route dependent on dynamin, Rac1 and Pak1.Virology 2010, 409:234–250.PubMedView Article
Agosto LM, Yu JJ, Liszewski MK, Baytop C, Korokhov N, Humeau LM, O’Doherty U: The CXCR4-tropic human immunodeficiency virus envelope promotes more-efficient gene delivery to resting CD4+ T cells than the vesicular stomatitis virus glycoprotein G envelope.J Virol 2009, 83:8153–8162.PubMedView Article
Bernstein BW, Bamburg JR: ADF/cofilin: a functional node in cell biology.Trends Cell Biol 2010, 20:187–195.PubMedView Article
Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K, Nishida E, Mizuno K: Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.Nature 1998, 393:809–812.PubMedView Article
Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA, Bernard O, Caroni P: Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase.Nature 1998, 393:805–809.PubMedView Article
Severson RK, Nomura AM, Grove JS, Stemmermann GN: A prospective study of demographics, diet, and prostate cancer among men of Japanese ancestry in Hawaii.Cancer Res 1989, 49:1857–1860.PubMed
Shimizu H, Ross RK, Bernstein L, Yatani R, Henderson BE, Mack TM: Cancers of the prostate and breast among Japanese and white immigrants in Los Angeles County.Br J Cancer 1991, 63:963–966.PubMedView Article
Adlercreutz H, Markkanen H, Watanabe S: Plasma concentrations of phyto-oestrogens in Japanese men.Lancet 1993, 342:1209–1210.PubMedView Article
Miltyk W, Craciunescu CN, Fischer L, Jeffcoat RA, Koch MA, Lopaczynski W, Mahoney C, Crowell J, Paglieri J, Zeisel SH: Lack of significant genotoxicity of purified soy isoflavones (genistein, daidzein, and glycitein) in 20 patients with prostate cancer.Am J Clin Nutr 2003, 77:875–882.PubMed
De Corte V, Gettemans J, Vandekerckhove J: Phosphatidylinositol 4,5-bisphosphate specifically stimulates PP60(c-src) catalyzed phosphorylation of gelsolin and related actin-binding proteins.FEBS Lett 1997, 401:191–196.PubMedView Article
De Corte V, Demol H, Goethals M, Van Damme J, Gettemans J, Vandekerckhove J: Identification of Tyr438 as the majorin vitroc-Src phosphorylation site in human gelsolin: a mass spectrometric approach.Protein Sci 1999, 8:234–241.PubMedView Article
Zhai L, Zhao P, Panebra A, Guerrerio AL, Khurana S: Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton.J Biol Chem 2001, 276:36163–36167.PubMedView Article
Bretscher A: Regulation of cortical structure by the ezrin-radixin-moesin protein family.Curr Opin Cell Biol 1999, 11:109–116.PubMedView Article
Huang C, Liu J, Haudenschild CC, Zhan X: The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells.J Biol Chem 1998, 273:25770–25776.PubMedView Article
Head JA, Jiang D, Li M, Zorn LJ, Schaefer EM, Parsons JT, Weed SA: Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton.Mol Biol Cell 2003, 14:3216–3229.PubMedView Article
Fan L, Di Ciano-Oliveira C, Weed SA, Craig AW, Greer PA, Rotstein OD, Kapus A: Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase.Biochem J 2004, 380:581–591.PubMedView Article
Chang F, Lemmon C, Lietha D, Eck M, Romer L: Tyrosine phosphorylation of Rac1: a role in regulation of cell spreading.PLoS One 2011, 6:e28587.PubMedView Article
Cory GO, Garg R, Cramer R, Ridley AJ: Phosphorylation of tyrosine 291 enhances the ability of WASp to stimulate actin polymerization and filopodium formation. Wiskott-Aldrich Syndrome protein.J Biol Chem 2002, 277:45115–45121.PubMedView Article
Pontow SE, Heyden NV, Wei S, Ratner L: Actin cytoskeletal reorganizations and coreceptor-mediated activation of rac during human immunodeficiency virus-induced cell fusion.J Virol 2004, 78:7138–7147.PubMedView Article
Costantino CM, Gupta A, Yewdall AW, Dale BM, Devi LA, Chen BK: Cannabinoid receptor 2-mediated attenuation of CXCR4-tropic HIV infection in primary CD4+ T cells.PLoS One 2012, 7:e33961.PubMedView Article
Kelly J, Beddall MH, Yu D, Iyer SR, Marsh JW, Wu Y: Human macrophages support persistent transcription from unintegrated HIV-1 DNA.Virology 2008, 372:300–312.PubMedView Article
Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.J Virol 1986, 59:284–291.PubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.