Activation of HIV-1 Long Terminal Repeat Transcription and Virus Replication via NF-κB-dependent and -independent Pathways by Potent Phosphotyrosine Phosphatase Inhibitors, the Peroxovanadium Compounds*

Replication of human immunodeficiency virus type 1 (HIV-1) is increased by different cytokines and T cell activators, also known to modulate tyrosine phosphorylation levels. A novel class of protein tyrosine phosphatase (PTP) inhibitors, peroxovanadium (pV) compounds, were tested for a putative effect on HIV-1 long terminal repeat (LTR) activity. We found that these PTP inhibitors markedly enhanced HIV-1 LTR activity in 1G5 cells, a stably transfected cell line that harbors an HIV-1 LTR-driven luciferase construct. A direct correlation between the extent of tyrosine phosphorylation and the level of HIV-1 LTR inducibility was seen after treatment with three different pV compounds. Transient transfection experiments were carried out in several T cell lines, and after addition of pV, a marked increase in HIV-1 LTR activity was measured. Monocytoid cells were tested using U937-derived cell lines and were also found to be sensitive to the pV-mediated potentiating effect on HIV-1 LTR activity. A significant reduction of the pV-mediated increase in HIV-1 LTR activity was seen in cells transiently transfected with an HIV-1 LTR-driven luciferase construct bearing a mutation in both NF-κB binding sites although detectable levels of induction remained. Electrophoretic mobility shift assays allowed the identification of the nuclear translocation of the NF-κB p50·p65 heterodimer complex induced by pV compounds. A dominant negative version of the repressor IκBα mutated on serines 32 and 36 impeded pV-induced NF-κB-dependent luciferase activity. Western blot analysis showed a clear diminution in the protein level of IκBα starting 30 min after pV treatment of Jurkat E6.1 cells which is indicative of its degradation. On the other hand, no increase in tyrosine phosphorylation was observed on IκBα itself. Finally, we tested the PTP inhibitors on four cell lines latently infected with HIV-1 and showed a consistent pV-mediated increase in virion production. Thus, our studies suggest that pV-mediated activation of HIV-1 LTR activity is controlled by the nuclear translocation of the NF-κB transcription factor, which is mediated by IκBα serine phosphorylation and degradation, but also by a still undefined NF-κB-independent pathway.


cells which is indicative of its degradation. On the other hand, no increase in tyrosine phosphorylation was observed on IB␣ itself. Finally, we tested the PTP inhibitors on four cell lines latently infected with HIV-1 and showed a consistent pV-mediated increase in virion production. Thus, our studies suggest that pV-mediated activation of HIV-1 LTR activity is controlled by the nuclear translocation of the NF-B transcription factor, which is mediated by IB␣ serine phosphorylation and degradation, but also by a still undefined NF-B-independent pathway.
One of the most important aspects of the life cycle of the human immunodeficiency virus type 1 (HIV-1) 1 is the modulation of its replication that is positively regulated by several cytokines or T cell activators (1). Most of these HIV-1 long terminal repeat (LTR) inducers act either completely or partially via the nuclear factor kappa B (NF-B) transcription factor. Although rarer, certain LTR activators are known to act through NF-B-independent pathways and include, for example, sodium butyrate, an inducer potentially acting on relaxation of chromatin by histone acetylation (2). NF-B has been shown to regulate viral transcription via the two NF-B sites located in the HIV-1 LTR enhancer region (3). The predominant form of this transcription factor has been identified as a heterodimer made of 50-kDa (p50) and 65-kDa (p65 or RelA) subunits (4,5). The heterodimeric NF-B complex is sequestered in the cytoplasm as an inactive precursor complexed with a repressor termed IB␣ that masks the nuclear localization signal of the heterodimer complex (6,7). Formation of the active nuclear form of NF-B composed of p50 and p65 subunits are induced after extra-or intracellular stimulation of cells. These stimulants appear to act primarily by inducing the release and degradation of IB␣ subsequent to its phosphorylation on both serine 32 and 36 residues (8), thereby allowing the rapid translocation of NF-B from the cytoplasm to the nucleus and binding on regulatory regions of genes bearing the NF-B binding sites (4,6,7). * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Ever since it became evident that activation of NF-B involves the release of the IB␣ repressor by direct phosphorylation, it has been attractive to evaluate the role of protein phosphorylation in directly or indirectly affecting the NF-B pathway. Overall, several protein kinases have been shown to be important in the regulation of NF-B translocation including protein kinase A, protein kinase C, Raf, and p59 fyn (9 -12), most of which have also been shown to modulate HIV-1 LTR activity. Further evidence supporting the role played by phosphorylation events in the control of NF-B activity came from the demonstration that okadaic acid, a selective inhibitor of Ser/Thr phosphatase (13), induces NF-B translocation in intact cells (14).
Tyrosine phosphorylation levels have been shown to often increase in signaling pathways leading to NF-B activation.
The level of tyrosine phosphorylation should thus be of importance in the control of the pathway(s) leading to the activation and translocation of NF-B. Tyrosine phosphorylation is regulated by two opposing enzymatic activities, namely protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP). These latter include a wide range of different enzymes, CD45 being the major constituent of T cell surface PTP (15). Modulation of intracellular phosphotyrosine content by PTP represents a crucial mechanism by which the cellular response is regulated. This fact is clearly demonstrated by studies using specific inhibitors against PTP that have been shown to stimulate T cell activation (16).
It is of prime importance to gain more knowledge of the molecular events controlling the transcriptional state of the HIV-1 proviral DNA. A large array of cellular factors have been shown to interact with binding sites located within the 5Ј regulatory region of the viral genome. Because an increase in both tyrosine phosphorylation and HIV-1 replication is seen following T cell activation, we were interested in seeing whether the induction of intracellular tyrosyl phosphorylation events, using new tyrosine phosphatase inhibitors, could modulate HIV-1 LTR activity. In the present study, we have made use of a set of pV compounds, which represent new potent structurally defined PTP inhibitors, and have demonstrated their capacity to markedly enhance HIV-1 LTR activity upon incubation with both T and monocytoid cells. Our results show that the induction is partly NF-B-dependent and requires serine phosphorylation of the IB␣ repressor.
These molecular constructs contain the luciferase reporter gene under the control of wild-type (GGGACTTTCC) or NF-B mutated (CT-CACTTTCC) HIV-1 LTR (28). The wild-type and dominant negative IB␣ expressing vectors pCMV-IB␣ and pCMV-IB␣ S32A/36A have been described previously (29). The pB-TATA-LUC contains the minimal HIV-1 B region and a TATA box placed upstream of the luciferase reporter gene (29). These three latter molecular constructs were generous gifts from Dr. W. C. Greene (The J. Gladstone Institutes, San Francisco). Carrier DNA (herring sperm DNA) was purchased from Sigma.
Preparation of pV Compounds-pV molecules were prepared as described previously (30). Briefly, V 2 O 5 was dissolved in an aqueous KOH solution and then mixed with 30% H 2 O 2 and the respective ancillary ligand in addition to the ethanol for optimal precipitation. Characterization of the pV molecules were carried out by infrared 1 H NMR and vanadium-51 ( 51 V) NMR spectroscopy. Stock solutions of pV molecules (1 mM in phosphate-buffered saline, pH 7.4) were kept at Ϫ20°C until used. Sodium orthovanadate (Sigma) was freshly dissolved in 10 mM HEPES, pH 7.4.
Transfection, Cell Treatments, and Luciferase and CAT Assays-To minimize variations in plasmid transfection efficiencies, transfected cells were pooled 24 h after transfection and were next separated into various treatment groups. Cells (5-10 ϫ 10 6 ) were first washed once in a TS buffer (25 mM Tris-HCl, pH 7.4, 5 mM KCl, 0.6 mM Na 2 HPO 4 , 0.5 mM MgCl 2 , and 0.7 mM CaCl 2 ) and resuspended in 0.5-1 ml of TS containing 15-30 g of the indicated plasmid(s) and 500 g/ml of DEAE-dextran (final concentration). The cells/TS/plasmid/DEAE-dextran mixture was incubated for 25 min at room temperature. Thereafter, cells were diluted at a concentration of 1 ϫ 10 6 /ml using complete culture medium supplemented with 100 M chloroquine (Sigma). After 45 min of incubation at 37°C, cells were centrifuged, resuspended in complete culture medium, and incubated at 37°C for 24 h. Transiently and stably transfected cells were seeded at a density of 10 5 cells per well (100 l) in 96-well flat-bottom plates. Cells were left untreated or were treated with the different peroxovanadium PTP inhibitors, bpV[HOpic], bpV[bipy], and bpV[pic] at specified concentrations (10 M in most experiments), phytohemagglutinin (PHA-P at 3 g/ml, Sigma), tumor necrosis factor-␣ (TNF-␣ at 2 ng/ml, R & D systems, Minneapolis), or phorbol myristic acid (PMA at 20 ng/ml, Sigma) in a final volume of 200 l. Next, these cells were incubated at 37°C for either 8 or 24 h. All experimental points were done in triplicate. Cell viability was estimated by the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay as described previously (31). Finally, luciferase or CAT activity was evaluated. Luciferase activity was determined following a modified version of a protocol (32). Briefly, following the incubation period, 100 l of cell-free supernatant were withdrawn from each well, and 25 l of cell culture lysis buffer (25 mM Tris phosphate, pH 7.8, 2 mM dithiothreitol, 1% Triton X-100, and 10% glycerol) were added before incubation at room temperature for 30 min. An aliquot of cell extract (20 l) was mixed with 100 l of luciferase assay buffer (20 mM Tricine, 1. Intracellular Flow Cytometry-Intracellular flow cytometry was performed as follows. Cells (5 ϫ 10 5 ) were washed once in PBS, pH 7.4, and were resuspended in 100 l of fresh complete culture medium before treatment with each peroxovanadium inhibitors (10 M) for 30 min at 37°C. Next, cells were washed once with PBS at room temperature and centrifuged 5 min at 300 ϫ g. Cell pellets were then fixed with 25 l of reagent A (Fix & Perm cell permeabilization kit from CALTAG Laboratories, South San Francisco, CA) and incubated 15 min at room temperature. Cells were washed in PBS, resuspended with 25 l of reagent B to which was added an anti-phosphotyrosine monoclonal antibody (clone 4G10; Upstate Biotechnology Inc., Lake Placid, NY) or a rabbit anti-Tat antibody (antiserum to HIV-1 Tat from Dr. Brian Cullen, AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH), vortexed gently, and incubated for 15 min at room temperature. Cells were subsequently washed with PBS 1% NaN 3 and resuspended with 100 l of PBS containing a fluorescein isothiocyanate-labeled goat anti-mouse or chicken anti-rabbit IgG antibody (1 g of total) and further incubated for 15 min at room temperature. Finally, cells were centrifuged and resuspended in 1% paraformaldehyde in PBS before being analyzed by flow cytometry (EPICS XL, Coulter Corp., Miami, FL). Cell surface CD4 expression was monitored as described previously (33).
Immunoblot Assay-Jurkat E6.1 (1 ϫ 10 6 cells/ml) were treated with the pV compound bpV[pic] (10 M) at various times and were resuspended in lysis buffer consisting of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 10% glycerol, 0.025 mM p-nitrophenyl guanidinobenzoate, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM sodium orthovanadate, and 10 mM sodium fluoride. The homogenate was vortexed and incubated for 45 min on ice with intermittent mixing before centrifugation to remove cellular debris. Protein content was determined by the commercial BCA Protein Assay Reagent (Pierce). Cleared cellular lysates or immunoprecipitated samples were electrophoresed on a 12% SDS-polyacrylamide gel electrophoresis, and the gel was transferred to a polyvinylidene difluoride membrane (Millipore, Mississauga, Ontario). Immunoblot detection was performed with either monoclonal anti-phosphotyrosine P-Tyr-02 (a kind gift from Dr. Vaclav Horejsi, Institute of Molecular Genetics, Prague, Czech Republic), 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), or PY20 (Transduction Laboratories, Lexington, KY) antibodies diluted 1:2000, or rabbit polyclonal antibodies specific for the C-terminal portion of IB␣ (kindly provided by Dr. W. C. Greene, The J. Gladstone Institute, San Francisco, CA) diluted 1:6000. Detection was carried out using either peroxidase-conjugated goat anti-mouse at a 1:20,000 dilution or peroxidase-conjugated sheep anti-rabbit antibodies at 1:10,000 dilution. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system according to the manufacturer's instructions (Amersham Corp.).
Immunoprecipitation Analyses-Jurkat E6.1 cells were treated at various times for up to 2 h with the pV compound bpV [pic]. Cleared cell lysates were first incubated with rabbit anti-IB␣ antibodies overnight at 4°C with intermittent mixing. Thereafter, protein G-Agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added and incubation at 4°C with intermittent mixing was pursued for 3 h. Immune complexes were washed three times in lysis buffer without Nonidet P-40 before analysis by immunoblot assay.
Preparation of Nuclear Extracts-1G5 cells were either left untreated or were incubated for different periods at 37°C with the peroxovanadium compounds (10 M). The incubation of 1G5 cells with the various stimulating agents was terminated by the addition of ice-cold PBS, and nuclear extracts were prepared according to the microscale preparation protocol (34). In brief, sedimented cells were resuspended in 400 l of cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride). After 10 min on ice, the lysate was vortexed for 10 s, and samples were centrifuged for 10 s at 12,000 ϫ g. The supernatant fraction was discarded, and the cell pellet was resuspended in 100 l of cold buffer B (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. Cellular debris was removed by centrifugation at 12,000 ϫ g for 2 min at 4°C, and the supernatant fraction was stored at Ϫ70°C until used.
Electrophoretic Mobility Shift Assay-Electrophoretic mobility shift assay was performed with 10 g of nuclear extracts. Protein concentrations were determined by the bicinchoninic assay with a commercial protein assay reagent (Pierce). Nuclear extracts were incubated for 30 min at 23°C in 15 l of buffer C (100 mM HEPES, pH 7.9, 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM dithiothreitol, 5 mM EDTA, 250 mM NaCl, 2 g of poly(dI-dC), 10 g of nuclease-free bovine serum albumin fraction V) containing 0.8 ng of 32 P-5Ј-end-labeled double-stranded DNA (dsDNA) oligonucleotide. Double-stranded DNA (100 ng) was labeled with [␥-32 P]ATP and T4 polynucleotide kinase in a kinase buffer (New England Biolabs, Beverly, MA). This mixture was incubated for 30 min at 37°C, and the reaction was stopped with 5 l of 0.2 M EDTA. The labeled oligonucleotide was extracted with phenol/chloroform and passed through a G-50 spin column. The dsDNA oligonucleotide, which was used as a probe or as a competitor, contained the consensus NF-B-binding site corresponding to the sequence 5Ј-ATGTGAGGG-GACTTTCCCAGGC-3Ј. A dsDNA oligonucleotide containing a mutated NF-B-binding site (in bold and underlined) was also used for competition (5Ј-ATGTGAGCTCACTTTCCCAGGC-3Ј). Oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). DNA⅐NF-B complexes were resolved from free labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels containing 50 mM Tris-HCl, pH 8.5, 200 mM glycine, and 1 mM EDTA. The gels were subsequently dried and autoradiographed. Cold competitor assays were carried out by adding a 100-fold molar excess of homologous unlabeled dsDNA NF-B oligonucleotide simultaneously with the labeled probe.
Supershift assays were performed by preincubation of nuclear extracts with 1 l of either preimmune serum (control) or specific antibodies in the presence of all the components of the binding reaction described above for 15 min at room temperature prior to the addition of the probe. Antibodies used are specific either to the p50 or p65 subunits of the NF-B complex and were kindly provided by Dr. Nancy Rice (NCI, Frederick, MD).
Reverse Transcriptase Assay-Reverse transcriptase activity was monitored as reported previously (33). Briefly, ACH-2, J1.1, OM10.1, and U1 cells were seeded at a density of 10 5  After incubation for 1 h at 37°C, samples were precipitated prior to filtration onto glass fiber filters by using a cell harvester system (Tomtec). The filters were dried, and radioactivity was measured in a liquid scintillation counter (1205/1204 BS Beta-plate; Wallac Oy, Turku, Finland). The assays were performed in triplicate.
Statistical Analyses-Statistically significant differences between groups were performed with the analysis of variance module of SAS software (version 6.07, SAS Institute, Cary, NC) using the Fisher least significant difference test. p values Ͻ0.05 were considered to be statistically significant (p values are given in the figure legends). All data are presented as mean Ϯ S.D.

RESULTS
Phosphorylation events have been shown to be important in several steps of the HIV-1 life cycle. This is probably associated with the fact that viral transcription is highly dependent on the intracellular environment (35). We were interested in testing whether PTP could directly modulate HIV-1 LTR activity. To achieve such a goal, the standard PTP inhibitor sodium vanadate tyrosine was used along with new pV compounds (30). In our studies, we have used several structurally defined pV compounds, each containing an oxo ligand, two peroxoanions, and an ancillary ligand located in the inner coordination sphere of vanadate.
Peroxovanadium Compounds Strongly Activate HIV-1 LTR Activity in T Cells-To assess whether PTP inhibitors could modulate HIV-1 LTR activity, pV compounds bpV[HOpic], bpV[bipy], and bpV[pic] were incubated for 24 h with 1G5 cells. This cell line is derived from the Jurkat E6.1 cell line and contains an integrated construct made of the luciferase reporter gene under the control of the HIV-1 LAI LTR. Sodium orthovanadate (Na 3 VO 4 ), a commonly used PTP inhibitor, was similarly tested in this series of experiments. As shown in Fig.  1, a weak increase in HIV-1-LTR-driven luciferase activity (2-fold) was obtained with 25 M of Na 3 VO 4 ( Fig. 1, panel A). The mitogenic agent PHA was used as a positive control and was shown, as expected, to induce HIV-1 LTR activity (4.6fold). However, the three pV derivatives used gave a significant increase in HIV-1 LTR-driven luciferase activity at a concentration of 10 M (Fig. 1, panels B-D). When positioned in order of importance, fold increments induced by the PTP inhibitors were bpV[bipy] (3.2-fold), bpV[HOpic] (5.2-fold), and bpV[pic] (8.1-fold). Although fold increase showed some degree of variation between experiments (data not shown), the order and the proportional difference between the fold increase induced by the three PTP inhibitors in the 1G5 cell line were constant. When lower concentrations of the pV compounds were used (0.1 and 1 M), no drastic HIV-1 LTR stimulation could be observed (Fig. 1). MTS assay for cell viability was performed in parallel and indicated no cytotoxic or cytostatic effect from the pV derivatives at 10 M concentration (data not shown). Thus, these results indicate that pV compounds (bpV[bipy], bpV[HOpic], and bpV[pic]) drastically increase HIV-1 LTR activity and to a much greater extent than Na 3 VO 4 .
Induction of HIV-1 LTR Activity by pV Derivatives Correlates with an Increase in Total Intracellular Phosphotyrosine Levels-To determine whether the difference in HIV-1 LTR activity correlated with the strength of the pV compounds to in-crease phosphotyrosine content, intracellular FACS analysis was performed with the anti-phosphotyrosine 4G10 monoclonal antibody. At concentrations lower than 10 M, no significant changes in the state of intracellular tyrosine phosphorylation in pV-treated 1G5 cells were apparent (data not shown). However, upon the addition of 10 M of bpV[bipy], bpV [HOpic] and bpV[pic], enhancement in phosphotyrosine contents was observed in 1G5 cells with mean fluorescence values (indicative of the number of molecules per single cell shown on a logarithmic scale) ranging from 2.7 to 8.37 (Fig. 2, panels A-C). To test whether a correlation existed between the increase in intracellular tyrosine phosphorylation and HIV-1 LTR activity, regression was performed between the luciferase activity measurements and FACS analysis mean value for each inhibitor. As seen in Fig. 2, panel D, a correlation between these two parameters could be clearly observed with a high significant value (r 2 ϭ 0.992). Thus, these results demonstrate that pV-mediated enhancement in the level of tyrosine-phosphorylated proteins is directly proportional to the observed up-regulation of HIV-1 LTR activity.
The Increase in HIV-1 LTR Activity by the pV Compounds Is Neither Cell-specific Nor HIV-1 LTR Strain-specific-To determine whether the induction of HIV-1 LTR activity by the pV compounds could be observed in other cell lines, transient transfection experiments were performed in several T cell lines using an HIV-1 LTR-driven reporter luciferase construct. After addition of any of the three pV derivatives at a concentration of 10 M, transfected Jurkat-tat cells showed a statistically significant induction of HIV-1 LTR-dependent reporter gene expression (p Ͻ 0.01) (Fig. 3, panel A). In these experiments, PHA was not found to be a good inducer of HIV-1 LTR activity as it was in the case of 1G5 cells because Jurkat-tat cells are negative for surface expression of the T cell receptor as monitored by FACS analysis (data not shown). Thus, PMA was used as a positive control and found to be a potent inducer of HIV-1 LTR activity (Fig. 3, panel A). We determined that a fraction of the pV-mediated stimulation of HIV-1 LTR transcription in Jurkat-tat cells was attributable to an increase in the level of endogenous trans-activating viral TAT protein as assessed by intracellular FACS analysis (data not shown). A statistically significant induction of HIV-1 LTR-driven luciferase activity (p Ͻ 0.01) was also seen following treatment with the three different PTP inhibitors of another T cell line (HPB-ALL) (Fig.  3, panel B). In addition, treatment with pV derivatives of M311, a Molt-4 derivative stably transfected with an HIV-1 LTR-CAT construct, or Molt-4 clone 8 transiently transfected with pLTRX-LUC led to a weak but statistically significant induction of HIV-1 LTR activity (1.6-to 1.7-fold over basal level) (data not shown). The parental Jurkat E6.1 cell line was shown to respond to a somewhat weaker extent than its stably transfected derivative 1G5 and Jurkat-tat cells. For example, a 1.6-, 1.8-, and 2.2-fold increase of HIV-1 LTR activity were detected following treatment of transiently transfected Jurkat E6.1 cells with bpV[bipy], bpV[HOpic], and bpV[pic], respectively (Fig. 3,  panel C). It should be noted that modulatory effects on HIV-1 LTR-dependent gene expression were until then all evaluated following a 24-h incubation period with pV compounds. A greater enhancement in HIV-1 LTR activity (3-8-fold) was seen when reporter gene expression was monitored at an earlier time post-treatment with pV derivatives (8 h) (Fig. 3, panel  D). Similar levels of induction in transiently transfected Jurkat E6.1 cells were observed with another HIV-1 pLTR-LUC construct (data not shown), which harbors an HIV-1 LTR promoter from a different genetic background (strain HXB2; molecular clone pLTR-LUC, see below). This demonstrates that pV-mediated activation of HIV-1 LTR activity is not restricted to a certain cell line nor to a particular HIV-1 LTR strain.
Induction of HIV-1 LTR Activity by pV Derivatives Is Partially Dependent on Intact NF-B Binding Sites-The NF-B transcription factor is a very potent factor and is often implicated in the induction of HIV-1 LTR activity (3). We were interested in testing the involvement of NF-B in the observed pV-mediated increase of HIV-1 LTR activity. Jurkat E6.1 cells were transfected with reporter gene plasmids possessing the full-length HIV-1 LTR promoter (pLTR-LUC) and the same HIV-1 LTR with mutated NF-B binding sites (pmBLTR-LUC) and then incubated with either PHA, TNF-␣, or the pV compounds for 8 h. As shown in Fig. 4, panel A, when pLTR-LUC was transfected in Jurkat E6.1, all activators, including the pV compounds, gave a marked increase in luciferase activity. As expected, no activation of HIV-1 LTR-driven gene activity was seen following treatment with PHA and TNF-␣ in Jurkat E6.1 cells transfected with pmBLTR-LUC (Fig. 4,  panel B). The increase in HIV-1 LTR-driven luciferase activity following treatment with PTP inhibitors was greatly diminished in cells transfected with the NF-B-mutated molecular construct but not totally abolished (Fig. 4, panel B). that induction of HIV-1 LTR activity by pV derivatives is partly NF-B-dependent.
pV Compounds Induce the Nuclear Translocation of NF-B-To further confirm the involvement of the transcription factor NF-B in the increase of the HIV-1 LTR activity by pV compounds, DNA mobility shift assays were carried out. First, we stimulated 1G5 cells for 1, 6, 12, and 24 h with the pV derivatives bpV[bipy] and bpV [pic]. Results demonstrated the presence of a specific band following treatment of 1G5 cells for 1 h, which was no longer observed after 6 h of treatment (data not shown). Further experiments revealed that all three PTP inhibitors at 1 h showed the presence of this signal (Fig. 5,  panel A, lanes 3-5). Competition assays with 100-fold excess of cold wild-type NF-B binding site showed complete disappearance of the signal for all nuclear extracts from pV-treated 1G5 cells (lanes 6 -8), whereas 100-fold excess of cold mutated NF-B oligonucleotide had no effect (data not shown). Data from this experiment clearly indicate that the binding was specific for NF-B.
Complexes binding to NF-B sequences may constitute a variety of hetero-or homodimers. These dimers can be composed of many distinct subunits, which are all members of the mammalian NF-B/rel homology domain family of transcription factors. To clearly identify the pV-induced NF-B complex, nuclear extracts from 1G5 treated with 10 M bpV[HOpic] were incubated with rabbit antibodies against the NF-B subunits p50 or p65 (Fig. 5, panel B). Upon addition of the polyclonal antibodies, the binding complex had completely disappeared (lanes 2 and 3), whereas a preimmune serum did not greatly affect the NF-B complex (lane 4). From these results, we conclude that translocation of the NF-B p50⅐p65 heterodimer is induced by the pV compounds and is responsible for the NF-B-dependent activation of the regulatory elements of HIV-1.

Serine Phosphorylation of the IB␣ Repressor Is Required for the pV-induced NF-B-mediated Activation of HIV-1 LTR Activity-NF-B
nuclear translocation is mainly mediated by the degradation of the repressor IB␣, which sequesters the complex in the cytoplasm (36 -38). This degradation is known to be almost completely dependent on the phosphorylation of the two serine residues 32 and 36, although tyrosine phosphorylation had been reported previously to act similarly (8,39). Because tyrosine phosphorylation is the targeted intracellular event in the presently studied HIV-1 LTR induction pathway, we wanted to verify if serine phosphorylation of IB␣ was involved in the pV-induced effect. To achieve this, we used a dominant negative version of IB␣ mutated to alanine on both serine 32 and 36 residues. The encoded protein is hence unable to be serine-phosphorylated but retains its ability to bind to the NF-B complex. In addition, a plasmid containing only the NF-B HIV-1 LTR binding sites upstream of a TATA box driving the expression of the luciferase gene (pB-TATA-LUC) was used in this set of experiments to exclusively study pV-induced activation of NF-B. Jurkat E6.1 cells were transfected with a 2 to 1 ratio of the pB-TATA-LUC and pCMV-IB␣ plasmids, the latter being either the wild-type (pCMV-IB␣ wild-type) or the dominant form of the repressor (pCMV-IB␣ serine 32/36). It should be noted that the pB-TATA-LUC plasmid resulted in a greater level of induction of luciferase activity (40 -50-fold increase) upon addition of the pV compounds as compared with those found with the pLTRX-LUC construct. This could be attributed to either the deletion of the HIV-1 LTR negative regulatory elements or to a better positioning of the NF-B binding sites for its trans-activating potential. Although transfection of the wild-type IB␣ had little effect on NF-B-induced reporter gene activity, when the mutated version of the IB␣ repressor was used, incubation of the cells with all three pV compounds showed an almost complete inhibition of NF-Bmediated luciferase activity (Fig. 6). TNF-␣, a positive control, was also similarly affected when IB␣ S32A/36A was added. These results indicate that pV-mediated activation and nuclear translocation of NF-B is resulting from the serine phosphorylation of IB␣ on residues 32 and 36.
No Changes in Tyrosine Phosphorylation of IB␣ Are Induced by pV Compounds-Since it has been previously reported that treatment of Jurkat E6.1 cells with pervanadate induced NF-B translocation via tyrosine phosphorylation of IB␣ with no concomitant degradation (40), we next wanted to evaluate whether a similar phenomenon was occurring with pV compounds. Results shown in Fig. 7 are indicative of no change in phosphotyrosine level of IB␣ throughout the time course using the anti-phosphotyrosine 4G10 or PY20 antibodies (panel A and data not shown). In comparison, at 30 and 60 min post-treatment with bpV[pic], a decrease in band intensity of the IB␣ protein was observable, which can likely be attributed to degradation of IB␣ (Fig. 7, panel B). Finally, total cell extracts from Jurkat E6.1 cells treated with bpV[pic] for similar amounts of time as shown in panels A and B were analyzed with an anti-phosphotyrosine antibody and demonstrated an increase in overall phosphotyrosine levels starting at the 5 min time point (Fig. 7, panel C). Our results thus suggest that pV molecules are triggering NF-B translocation by a degradation process of the IB␣ repressor which is dependent on intracellular tyrosine phosphorylation levels but with no concomitant increase in tyrosine phosphorylation of IB␣ itself.
pV Compounds Stimulate Viral Production in T Cell Lines Latently Infected with HIV-1-Since pV compounds are able to increase HIV-1 LTR activity, we next wanted to test their potency in the context of an entire viral genome. This is based on the observation that HIV-1 replication is a complex process regulated by proteins of viral and cellular origins that have been shown to work in cis and trans. As presented in Fig. 8, treatment of J1.1, a cell line latently infected with HIV-1, with all three inhibitors induced HIV-1 replication over time as determined by an increase in reverse transcriptase activity. PMA was used as a positive control and also induced HIV-1 replication (data not shown). Another T cell line containing latently integrated HIV-1 DNA, ACH-2, gave similar induction of reverse transcriptase activity when two of the three inhibitors (bpV[HOpic] and bpV[pic]) were tested (data not shown).
In conclusion, pV compounds can also stimulate HIV-1 replication in latently infected T cells.
pV Derivatives Increase HIV-1 LTR Activity and Virion Production in Monocytoid Cells-Monocyte-derived macrophages are considered to be the most frequently identified hosts of HIV-1 in tissues of infected individuals (41). We therefore tested the effect of pV molecules on U38, a U937-derived cell line stably transfected with an HIV-1 LTR-CAT construct. After addition of the three inhibitors to this cell line, CAT activity was drastically increased (up to 18-fold) as compared with untreated cells (Fig. 9, panel A). A more modest but significant increase in CAT activity was also seen with PMA (data not shown). To further test the induction of HIV-1 LTR activity in cells of monocytoid origin, U1 cell line, which contains two integrated full-length proviral DNA copies, was incubated with the PTP inhibitors. Again, a time-dependent increase in reverse transcriptase activity was observed for all pV compounds tested (Fig. 9, panel B). Similar results were obtained when the latently infected monocytoid OM10.1 cell line was tested with the inhibitors bpV[HOpic] and bpV[pic] (data not shown). The data presented above hence indicate that pV molecules can increase HIV-1 LTR activity and virus replication in a wide range of cell types including monocytoid cell lines. DISCUSSION HIV-1 replication is controlled by several different cytokines and mitogens. Although several reports have focused on the agents stimulating or repressing LTR activity, little information regarding intracellular pathways that act on HIV-1 LTR regulation is available. Most information is centered around the NF-B pathway, which is dependent on the degradation of the IB␣ repressor induced by the phosphorylation of 2 serine residues (8). Although central to many different pathways, tyrosine phosphorylation events per se have not been directly investigated in HIV-1 regulation. In this study, we have thus analyzed the role of tyrosine phosphorylation on HIV-1 LTR regulation. pV compounds, previously characterized as potent PTP inhibitors, were used in this study to increase intracellular phosphotyrosine levels.
Our results first demonstrated that pV compounds were effective in inducing HIV-1 LTR activity in 1G5 T lymphoid cells and to a much higher extent than sodium orthovanadate, which is consistent with the reported observation showing that pV molecules are more potent PTP inhibitors than sodium orthovanadate (30). Our results are also in agreement with a previous study that reported an activation of HIV-1 LTR activity in 1G5 cells following treatment of vanadate and hydrogen peroxide (42), a combination known to form pervanadate mol- ecules when intermixed. However, our findings are different from a previous study that has shown that pervanadate and phenylarsine oxide, two PTP inhibitors, can abrogate TNF-␣mediated NF-B translocation (43). This discrepancy might be explained by the targeting of different PTP in the two different pathways analyzed. Indeed, the different pV compounds used in the present work vary in terms of their ancillary ligand which is attached to the vanadate core. This may give a different specificity to these molecules in terms of the PTP affected. This notion is exemplified by previous results showing that pervanadate and vanadate demonstrate distinct potency and substrate specificity (44). However, it should be noted that our data showed a strong correlation between the level of intracellular tyrosine phosphorylation and the induction of HIV-1 LTR activity. This most likely suggests that a similar or a set of similar PTP are targeted by the pV derivatives but to a different extent.
We have determined that pV compounds induced HIV-1 LTR activity in several lymphoblastoid T and monocytoid cells. The pV-mediated increases in HIV-1 LTR transcription were greater in stably transfected cells than in transient transfection experiments. Although cell type might explain some variation, it is likely that the number of cells carrying the molecular HIV-1 construct, as well as differences in the number of copies introduced per cell, might be responsible for the observed variations in the response to pV derivatives. The effects of pV molecules in transiently transfected Jurkat E6.1 cells were measured after 8 h of treatment, which permitted higher levels of enhancement as compared with the 24-h treatment period. However, a more significant increase in HIV-1 LTR transcription was seen following treatment for 24 h of HPB-ALL with the pV compounds when compared with Jurkat E6.1 (Fig. 3, panel B), indicating variation in responsiveness to pV among the different T cell lines.
Based on DNA mobility shift assays and transient transfection experiments with HIV-1 LTR constructs bearing mutated NF-B binding sites, our results suggest that part of the pVinduced effect on HIV-1 LTR activity is mediated via the activation and nuclear translocation of NF-B. This is similar to most HIV-1 LTR activators which stimulate LTR activity via this transcription factor. Although several forms of NF-B dimers exist, our supershift assays are indicative of p50⅐p65 heterodimer being the bound component of the activated complex. This dimer is in fact the most important activator of all formed heterodimers related to the rel family (45). These results are in agreement with the fact that several pathways inducing NF-B translocation have been known to act through an increase in tyrosine phosphorylation. For example, it has been shown that overexpression of p59 fyn in T cell lines could stimulate HIV-1 LTR activity by NF-B-like DNA binding proteins (9). We have further analyzed the pathway by which NF-B translocation is induced by pV inhibitors. It is known that the IB␣ repressor sequesters the NF-B complex in the cytoplasm by an interaction with the nuclear localization signal region. Upon proper activation, serine phosphorylation of residues 32 and 36 occurs on the IB␣ repressor by a kinase whose activity is dependent on ubiquitination (46). Thus, phosphorylation targets IB␣ repressor to the proteasome via a set of entrained ubiquitination events (8,47). On the other hand, it has recently been reported that tyrosine phosphorylation of IB␣ in hypoxia-treated Jurkat cells could lead to activation of NF-B (39). Our results indicate that pV compounds induce NF-B-dependent activation of HIV-1 LTR activity via serine phosphorylation events since a dominant negative version mutated at serine 32 and 36 abrogated the pV-mediated effect on HIV-1 transcription. These results are again similar to NF-B translocation induced by several T cell activation signals (i.e. TNF-␣, PMA, lipopolysaccharide) which all act through serine phosphorylation of IB␣ resulting in its concomitant degradation (48).
Our Western analyses confirmed the importance played by serine phosphorylation since degradation was apparent in Jurkat E6.1 cells treated with the pV compound bpV[pic] with no indication of stimulated tyrosine phosphorylation of IB␣. Our observations are thus in sharp contrast with a recently described work which demonstrated that pervanadate can lead to translocation of NF-B via tyrosine phosphorylation of IB␣ with no concomitant proteolytic degradation of IB␣ (40). This discrepancy might be related to the possibility that pV compounds might, as postulated above, target different PTP. In agreement with this hypothesis, we found that H7, a protein kinase C inhibitor, could drastically reduce pV-induced activation of HIV-1 LTR activity (data not shown), while such a treatment had no effect on NF-B translocation mediated by pervanadate (40).
The participation of phosphatase(s) in the activation of NF-B was previously provided by a study demonstrating that the Ser/Thr PTP inhibitor okadaic acid is also a potent activator of NF-B (49). Our results represent the first demonstration that specific inhibitors of PTP lead to NF-B activation via a site-specific serine phosphorylation of IB␣. Thus, our studies imply that the serine phosphorylation state of IB␣ is controlled by the dynamic balance between PTK and PTP activities. It is also indicative of the presence of a common kinase(s) activated by several different signaling pathways that is ultimately participating to the regulation and function of NF-B. PTP could be a very important regulator of NF-B translocation keeping the activating pathway at a resting state. This is reminiscent of the implication of certain PTP, such as CD45 and PTP-1C, in restraining other activated pathways by terminating tyrosine phosphorylation signaling (50,51). Thus, the present data highlight a potential role for PTP as negative regulatory elements in NF-B activation.
pV inhibitors were also found to stimulate HIV-1 production in four different cell lines latently infected with HIV-1 which is indicative of a common signaling pathway for the induction of NF-B translocation in several different cell lines that ultimately leads to HIV-1 production regardless of the cause of viral latency (52,53). It has been shown that the frequency of cells carrying transcriptionally active HIV-1 proviral DNA in infected individuals is at least 1 to 2 orders of magnitude lower than the total amount of infected cells (54), indicating that the majority of infected cells exist in a state of transcriptional latency. Thus, we can postulate that PTP are implicated to some extent in the regulation of HIV-1 expression by keeping the virus under some form of latency.
We have demonstrated that the pV compounds induce the HIV-1 LTR activity through two distinct pathways, one which is NF-B-dependent and one which is NF-B-independent. The NF-B-independent pathway remains to be elucidated, but preliminary results from our laboratory suggest that this mode of activation is not exclusive to the HIV-1 LTR (data not shown). A similar bimodal NF-B-dependent and -independent type of HIV-1 LTR induction has also been reported with okadaic acid (49). It is interesting to note that this inhibitor acts on serine/ threonine phosphatases and might thus trigger a cascade similar, at some point, to that initiated by pV compounds.
The most important question resulting from our findings is the identification of the targeted PTP and participating intracellular second messengers. Although several different PTPs have been described to date, the integral membrane PTP CD45 seems a good candidate for many reasons. This phosphatase is the most abundant leukocyte cell surface glycoprotein and is present in all hematopoietic cell types (15). It is known to be important in T cell activation via the activation of the protein tyrosine kinases p56 lck and p59 fyn (15,55). Treatment with pervanadate was demonstrated to inhibit most of the CD45 PTP activity (16) and to affect CD45 interaction with other proteins including p56 lck (56). More importantly, in CD45 negative cell lines, HIV-1 LTR activity is greater than in the wild-type counterpart due to constitutive translocation of NF-B (57). Studies are currently in progress to investigate the role played by CD45 in pV-mediated activation of HIV-1 LTR transcription.
Our results suggest for the first time that a still undefined protein tyrosine phosphatase(s) is a key element in the signal transduction pathway leading to IB␣ serine phosphorylation and NF-B nuclear translocation. In light of our results, we hypothesize two possible modes of action by which pV com-pounds could lead to IB␣ serine phosphorylation. First, it is conceivable that an IB␣-specific serine/threonine kinase, part of the presumed multisubunit kinase complex (46), might be activated by tyrosine phosphorylation which, in turn, would be controlled by a pV-sensitive PTP. Second, pV compounds might be inactivating a dual-specificity phosphatase resulting in an increase of the half-life of IB␣ serine phosphorylation. This eventuality is based on the postulate that IB␣ phosphorylation level is controlled by an equilibrium between PTK and PTP activities.
In conclusion, these pV derivatives may allow us to further characterize the signaling events that contribute to activation of NF-B following serine phosphorylation of IB␣. Moreover, pV compounds may also permit us to acquire a better understanding of the intracellular factor(s) that keep the integrated HIV-1 genome under a certain latent state. Identification of PTP involved in serine phosphorylation of IB␣ by pV molecules may provide new opportunities to develop therapeutic strategies aimed at inhibiting dysregulated NF-B functions seen in certain diseases.