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Originally published online as doi:10.1189/jlb.1205723 on July 7, 2006

Published online before print July 7, 2006
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(Journal of Leukocyte Biology. 2006;80:640-650.)
© 2006 by Society for Leukocyte Biology

AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation

Christian Grant*,{dagger}, Pooja Jain{dagger}, Michael Nonnemacher{dagger}, Katherine E. Flaig{dagger}, Bryan Irish{dagger}, Jaya Ahuja{dagger}, Aikaterini Alexaki{dagger}, Timothy Alefantis{ddagger} and Brian Wigdahl{dagger},1

* Viral Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland;
{dagger} Department of Microbiology and Immunology, and Center for Molecular Virology and Neuroimmunology, Center for Cancer Biology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, Pennsylvania; and
{ddagger} Vital Probes, Inc., Mayfield, Pennsylvania

1 Correspondence: Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129. E-mail: bwigdahl{at}drexelmed.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human T cell leukemia virus type 1 (HTLV-1) has previously been shown to infect antigen-presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV-1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV-1 viral gene expression in established monocytic cell lines was examined. U-937 promonocytic cells transiently transfected with a HTLV-1 long-terminal repeat (LTR) luciferase construct were treated with phorbol 12-myristate 13-acetate (PMA) to induce cellular differentiation. PMA-induced cellular differentiation resulted in activation of basal and Tax-mediated transactivation of the HTLV-1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA-induced cellular differentiation induced DNA-binding activity of cellular transcription factors to Tax-responsive element 1 (TRE-1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP-1) family of basic region/leucine zipper proteins (Fra-1, Fra-2, JunB, and JunD) were induced to bind to TRE-1 repeat II during cellular differentiation. Inhibition of AP-1 DNA-binding activity by overexpression of a dominant-negative c-Fos mutant (A-Fos) in transient expression analyses resulted in severely decreased levels of HTLV-1 LTR activation in PMA-induced U-937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV-1 viral gene expression may become up-regulated by AP-1 during differentiation into macrophages or dendritic cells.

Key Words: HTLV-1 • LTR • Tax • monocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM), also referred to as tropical spastic paraparesis (TSP) [1 2 3 4 ]. However, only ~5% of individuals infected with HTLV-1 develop ATL or HAM/TSP, and ~95% remain asymptomatic carriers. It has been reported that the proviral DNA load and the viral RNA load are critical factors promoting disease progression in infected individuals [5 6 7 ]. HTLV-1 gene expression from the viral promoter contained within an integrated provirus is regulated at the transcriptional level by numerous cellular transcription factors, including activating transcription factor 1 (ATF-1), ATF-2, cyclic adenosine monophosphate (cAMP)-responsive element-binding protein 1 (CREB-1), CREB-2 (also known as ATF-4), cAMP-responsive element (CRE) modulator, activator protein 1 (AP-1), c-Ets, c-Myb, Gli-2, specificity protein 1 (Sp1), and Sp3 [8 9 10 11 12 13 14 15 16 ]. Cellular factors, together with the coactivators CREB-binding protein (CBP)/p300, regulate low levels of basal viral gene expression [17 18 19 20 ]. Synthesis of the viral oncoprotein Tax within infected cells and subsequent nuclear localization of this protein result in transactivation of viral gene expression by Tax through direct interactions with the basic region and leucine zipper (bZIP) of members of the ATF/CREB family, which leads to the stabilization of these dimers and enhanced DNA binding of the ATF/CREB-Tax complex to the 21-base pair repeats within Tax-responsive element 1 (TRE-1) [21 ]. Each TRE-1 repeat consists of a core viral CRE, which facilitates binding by ATF/CREB factors and flanking G/C-rich sequences. Tax makes direct contacts with the GC-rich sequences and has been shown to stabilize ATF/CREB-Tax complexes on the viral promoter [17 , 22 ]. Tax has been shown to recruit CBP/p300 to the viral promoter, and the intrinsic histone acetyltransferase activity of CBP/p300 has been shown to facilitate localized nucleosomal histone acetylation and remodeling of the chromatin environment, resulting in recruitment of RNA polymerase II and general transcription factors and coordinated high-level HTLV-1 gene expression [23 , 24 ]. In addition to its activity as an oncoprotein [25 ], Tax has been shown to be present in the serum and colony-stimulating factor of HAM/TSP patients in cell-free form [26 ]. However, it is not clear yet whether cell-free Tax was the result of apoptosis or necrosis of HTLV-1-infected cells or if it was secreted from selected infected cell populations. Consistent with the concept of Tax secretion, we have previously reported the presence of a nuclear export signal within Tax [27 ] and the secretion of full-length Tax from transfected cells [28 ]. Cytoplasmic localization of Tax could have profound effects on the ability of this protein to interact with cellular transcription factors.

The primary cellular target of HTLV-1 has been shown to be CD4+ T lymphocytes, and this cell population represents a major viral reservoir within infected individuals [7 , 29 ]. HTLV-1 has also been shown to infect CD8+ T cells, B cells, monocytes, dendritic cells (DCs), microglial cells, and astrocytes in vivo, although to a much lesser extent [18 , 30 , 31 ]. As initiators of innate and adaptive antiviral immune responses, antigen-presenting cells (APCs) and their precursors (including monocytes, macrophages, and DCs) play an important role in protecting the host from infectious pathogens [32 33 34 35 ]. It is interesting that previous results have demonstrated that in vitro spontaneous proliferation of CD4+ and CD8+ T cells from the peripheral blood of HAM/TSP patients could be abrogated by the depletion of DCs from the ex vivo cultures [36 , 37 ]. These observations have led us to hypothesize that HTLV-1 infection and viral gene expression within APCs, followed by presentation of viral antigenic peptides on the cell surface, promote (at least in part) the highly aggressive HTLV-1-specific immune response detected in patients with HAM/TSP. We have recently shown that Tax induces the expression of DC markers associated with maturation and activation using a murine DC line [38 ] and primary human monocyte-derived DCs [39 ]. As basal and activated HTLV-1 gene expression has been shown to be heavily dependent on cellular transcription factors, their expression profile within infected monocytes and DCs may be critically involved in the control of viral gene expression within these pathogenically relevant cell populations. In this study, we have initiated investigations to examine the transcriptional activation of the HTLV-1 long-terminal repeat (LTR) within cells of the monocyte/macrophage lineage and the molecular mechanism(s) regulating viral gene expression during differentiation into mature APCs.

Previous studies have implicated AP-1 as a critical regulator of monocytic differentiation [40 41 42 43 44 ]. AP-1 is a family of bZIP transcription factors (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2), which function as dimers (Jun:Jun homodimers/heterodimers or Fos:Jun heterodimers) and are involved in the regulation of several myeloid-specific genes (e.g., CD16, macrophage scavenger receptor, lysozyme, macrophage mannose receptor, and PU.1) [45 ]. Although the expression of AP-1 family members can be detected in many different cell types [46 ], AP-1 expression and DNA-binding activity have been shown to be up-regulated during monocytic differentiation [40 , 47 ]. Sequence-specific DNA-binding activity of AP-1 has been shown to exhibit high affinity for 12-O-tetradecenoylphorbol-13-acetate (TPA)-responsive elements in which the consensus sequence (TGAC/GTCA) is nearly identical to the CRE consensus sequence (TGACGTCA), AP-1 and ATF/CREB factors are capable of physically competing with each other for binding to and regulating transcriptional activation from promoter elements [48 , 49 ]. This form of transcriptional cross-talk is not limited to just cellular promoters but has also been shown to involve the HTLV-1 LTR [15 , 50 51 52 53 ]. Overexpression of the viral oncogene v-Jun (derived from avian sarcoma virus 17), c-Jun alone, or c-Fos in combination with c-Jun has previously been shown to up-regulate basal activation of the HTLV-1 LTR [50 , 51 ]. Recently, a novel viral protein HTLV-1 bZIP factor, HBZ, has been shown to interact with c-Jun and repress c-Jun-mediated transcription by abrogating its DNA-binding activity [54 ]. AP-1-mediated activation of the HTLV-1 LTR resulted from direct interactions formed between AP-1 and TRE-1 repeat II [51 , 53 ]. AP-1 expression and DNA-binding activity have been shown to be up-regulated during monocytic differentiation and T cell activation by stimulation of the protein kinase C (PKC) pathway [40 , 55 ]. It is interesting that PKC-mediated activation of HTLV-1 gene expression has been shown to be dependent on TRE-1 repeat II [56 ]. These observations suggest that monocytic differentiation-induced activation of PKC may activate AP-1 expression and DNA-binding activity, resulting in up-regulation of basal LTR activity. Additional studies demonstrating that Tax can also up-regulate AP-1 expression and DNA-binding activity have suggested that Tax and AP-1 may function synergistically to transactivate HTLV-1 viral gene expression [50 , 57 , 58 ]. We demonstrate herein that AP-1 up-regulates HTLV-1 LTR activation during phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation. Treatment of myeloid lineage precursor cells (U-937, an immature, monocytic cell line) with PMA resulted in up-regulation of CD14 and CD11b expression, consistent with the ability of PMA to induce terminal monocytic differentiation of several myeloid leukemia cell lines [10 , 59 , 60 ]. The effects of PMA and Tax were synergistic with respect to transactivation of the LTR. Electrophoretic mobility shift (EMS) analyses using nuclear extracts derived from U-937 cells demonstrated that PMA-induced monocytic differentiation resulted in enhancement of TRE-1 repeat II-specific DNA-protein complex formation. The TRE-1 repeat II-specific DNA-protein complexes consisted of the AP-1 components Fra-1, Fra-2, JunB, and JunD, as demonstrated by supershift analyses (although the major component was JunB). Finally, overexpression of a dominant-negative AP-1 factor (A-Fos) in U-937 cells completely abrogated PMA-induced basal activation of the HTLV-1 LTR. Collectively, these studies suggest that low-level HTLV-1 viral gene expression within cells of the myeloid lineage may become up-regulated during differentiation toward functional APCs and may contribute to the persistent activation of HTLV-1-specific CD4+ and CD8+ T cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human immature monocytic U-937 cells (ATCC, CRL-1593.2) and human histiocytic lymphoma TUR (TPA-U-937-resistant) cells (ATCC, CRL-2367) were cultured and maintained in RPMI-1640 (Mediatech, Herndon, VA) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), sodium bicarbonate (0.3%, Mediatech), and 10% fetal bovine serum (FBS; HyClone, Logan, UT). As one approach to induce monocytic differentiation, PMA was added directly to the cell culture media of U-937 (500 nM, Sigma Chemical Co., St. Louis, MO) cells for 24–48 h. TUR cells were differentiated with 1,25-dihydroxyvitamin D3 (VD3; 0.5–1 µm, Sigma Chemical Co.), added directly to culture medium of TUR cells for 24–48 h, as they exhibit resistance to PMA-induced differentiation.

Fluorescent-activated cell sorting (FACS)
U-937 and TUR cells (1x106 cells/mL) were treated with PMA (500 nM) or VD3 (500 nM) for 24–48 h. Unstimulated or treated cells were incubated with 20 µL of the appropriate antibody (50 ng/µl) or isotype control (eBiosciences, San Diego, CA) for 30 min in 100 µL FACS buffer [1x Dulbecco’s phosphate-buffered saline (DPBS; Mediatech), NaN3 (0.05%), FBS (2%)] at 4°C. Cells were washed once with ice-cold FACS buffer, washed again with ice-cold 1x DPBS, and resuspended in 400 µL paraformaldehyde (2%). Flow cytometric analysis was performed with a FACScan 2000, and data were analyzed with FlowJo software (Tree Star, San Carlos, CA).

Nuclear extract preparation and EMS analyses
Small-scale nuclear extracts were prepared from low-passage, exponentially growing cells. Briefly, cells (1x107) were collected by centrifugation, washed once with 1x DPBS, and lysed in ice-cold lysis buffer HEPES (10 mM), pH 7.9, KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), IGEPAL (0.4%), dithiothreitol (DTT; 1 mM), phenylmethylsulfonyl fluoride (PMSF; 0.5 mM), and HALT protease inhibitor cocktail (Pierce, Rockford, IL; 1:100). After centrifugation (1000 g), the supernatant (cytoplasmic extract) was discarded. The pelleted nuclei were resuspended gently in nuclear extract buffer [HEPES (20 mM), NaCl (0.4 M), EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF (1 mM), and HALT protease inhibitor cocktail (1:100, Pierce)], shaken vigorously for 30 min at 4°C, and subjected to centrifugation for 10 min (16,000 g). The supernatant (nuclear extract) was transferred to a new Eppendorf tube on ice, frozen in liquid nitrogen, and stored at –80°C. The protein concentration of each sample was determined by Bradford assay as described by the manufacturer (Bio-Rad, Hercules, CA).

The following sequences were used to generate double-stranded, gel-purified oligonucleotides for EMS analysis: 5'-AGACTAAGGCTCTGACGTCTCCCCCCAGAGG-3' and 5'-CCTCTGGGGGGAGACGTCAGAGCCTTAGTCT-3' (TRE-1 repeat I); 5'-CAGGCTAGGCCCTGACGTGTCCCCCTGAAGA-3' and 5'-TCTTCAGGGGGACACGTCAGGGCCTAGCCTG-3' (TRE-1 repeat II); 5'-CAGGCTAGGCCCTGACTAATCCCCCTGAAGA-3' and 5'-TCTTCAGGGGGATTAGTCAGGGCCTAGCCTG-3' (MII 12,13,14 GTG:TAA); 5'-GCCCTCAGGCGTTGACGACAACCCCTCACCT-3' and 5'-AGGTGAGGGGTTGTCGTCAACGCCTGAGGGC-3' (TRE-1 repeat III); 5'-AGACCTCCGGGAAGCCACCGGGAACCACCCATTTCCTCCCCATGTTT-3' and 5'-AAACATGGGGAGGAAATGGGTGGTTCCCGGTGGCTTCCCGGAGGTCT-3' (TRE-2); 5'-TACAGGCATACCGGTTCCGTAGTGA-3' and 5'-TCACTACGGAACCGTTATGCCTGTA-3' (c-Myb consensus sequence); 5'-AGTTGAGGGGACTTTCCCAGGC-3' and 5'-GCCTGGGAAAGTCCCCTCAACT-3' [nuclear factor-{kappa}B (NF-{kappa}B) consensus sequence]; 5'-ATTCGATCGGGGCGGGGCGAGC-3' and 5'-GCTCGCCCCGCCCCGATCGAAT-3' (Sp1/Sp3 consensus sequence); and 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' and 5'-CTAGCTCTCTGACGTCAGGCAATCTCT-3' (CREB consensus sequence). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA), annealed by heating at 95°C for 5 min, gradually cooled to room temperature, and gel-purified. Radiolabeling of probes was performed by an end-labeling reaction with T4 DNA kinase (Promega, Madison, WI) and [{gamma}-32P]adenosine 5'-triphosphate. Labeled probes (75,000 counts per minute) were incubated in reaction buffer containing dialysis buffer [HEPES (25 mM), pH 7.9, glycerol (10%), KCl (100 mM), EDTA (0.1 mM)], poly (dI-dC), 2 µg, and nuclear extract (6 µg)] for 30 min at 30°C. DNA-protein complexes were then resolved on a 5% polyacrylamide gel at 200 V in 1x Tris-boris acid-EDTA buffer. The gels were dried under vacuum at 80°C for 1.5 h prior to autoradiography. For supershift analysis, 2 µL of the appropriate polyclonal antibody (Santa Cruz Biotechnologies, CA) was added during the final 15 min of the 30°C incubation.

Plasmid construction
pGL3-based HTLV-1 LTR luciferase reporter vector (pU3R-luc) was used as a template to generate a HTLV-1 minimal promoter (pU3R{Delta}-77-luc), where sequences upstream of the TATAA box had been removed by direct polymerase chain reaction amplification of the desired regions of the HTLV-1 LTR (–77 to +268). The amplification products were digested and cloned into the pGL3-basic luciferase reporter vector (Promega). The content of the mutant pU3R-luc construct was confirmed by automated DNA sequencing and sequence analysis using Lasergene software (DNASTAR, Inc., Madison, WI).

Transient transfections
Exponentially growing U-937 or TUR cells were plated onto six-well tissue-culture plates on the day of transfection at a concentration of 1 x 106 cells/well. Transient transfections were performed using the FuGene6 transfection reagent as described by the manufacturer (Roche, Indianapolis, IN). A HTLV-1 LTR luciferase reporter construct (pU3R-luc) or a HTLV-1 minimal promoter missing sequences upstream of the TATAA box (pU3R{Delta}-77-luc) was transfected alone or cotransfected together with the appropriate expression vectors: plasmid cytomegalovirus (pCMV)-Tax, pCMV-c-Jun, pCMV-CREB-1, A-Fos, and/or A-CREB. pCMV-Tax has been described previously [61 ]. pCMV-c-Jun was provided by Dr. Shao-Cong Sun (Pennsylvania State College of Medicine, Hershey). pCMV-CREB-1 was purchased from BD Biosciences (San Jose, CA). Dominant-negative AP-1 (A-Fos) and ATF/CREB (A-CREB) expression vectors were kindly provided by Dr. Charles Vinson (National Institutes of Health, Bethesda, MD). pUC18 plasmid DNA was used to give each reaction within an experiment an equal amount of total DNA. Cells were harvested 24 h and 48 h post-transfection, and cell lysates were prepared using 50 µL 1x passive lysis buffer (Promega). In some experiments, PMA or VD3 (as indicated) was added to the reactions 30 min post-transfection. Luciferase activity was assayed using the dual luciferase assay system as described by the manufacturer (Promega). Normalization to an internal control plasmid was not performed as a result of several studies reported previously, demonstrating the responsiveness of widely used, internal reference reporters to cotransfected transcriptional regulators (e.g., HTLV-1 Tax) [62 63 64 65 ].

Statistical analysis
Data collected for transient transfections were analyzed for statistical significance using JMP software Version 5.1.1 (SAS Institute, Cary, NC) Briefly, datasets from each experiment were imported into JMP, and an ANOVA was performed. The statistical significance of each data set within an experiment was determined using the Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PMA-induced monocytic differentiation stimulates HTLV-1 LTR activation
HTLV-1 has been shown to infect peripheral blood monocytes and DCs in vitro and in vivo [30 , 66 ]. However, little information is available concerning HTLV-1 viral gene expression during differentiation of infected myeloid precursors. Previous reports have demonstrated the use of U-937 cells for studying cellular gene expression associated with promonocyte to macrophage differentiation [41 , 59 , 60 , 67 ]. To establish the use of these cells for analysis of HTLV-1 LTR activation during monocytic differentiation, FACS analysis of U-937 cells in the absence or presence of PMA for 24 h was performed. Results shown in Figure 1 illustrate that expression of CD11b and CD11c (myeloid lineage-specific adhesion molecules), CD14 (a macrophage-specific lipopolysaccharide coreceptor), and CD69 (an early activation marker) is up-regulated in PMA-treated U-937 cells. Down-regulation of CD4 expression was a specific effect of PMA and not related to monocytic differentiation, as previous studies have demonstrated this effect in a number of other cell populations. These results have suggested that PMA treatment of U-937 cells promoted phenotypic monocytic differentiation similar to that observed during in vitro differentiation of primary monocyte-derived macrophages [68 , 69 ].


Figure 1
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  		Figure 1. U-937 monocytic cells can be induced to differentiate by PMA. U-937 monocytic cells were left untreated (thin, gray lines) or treated with PMA for 24 h (bold, black lines). Cell surface markers associated with monocytic differentiation (CD11b, CD11c, CD14, CD69, CD4) were analyzed by FACS analysis. The relative level of expression is indicated on the x-axis, and the number of events (cell counts) is indicated on the y-axis.

 
To determine whether HTLV-1 LTR activation could be affected by monocytic differentiation, U-937 cells were transiently transfected with a HTLV-1 LTR luciferase reporter vector (pU3R-luc) or a HTLV-1 minimal promoter in which sequences upstream of the TATAA box have been deleted (pU3R{Delta}-77-luc) in the absence or presence of an expression vector encoding HTLV-1 Tax (pCMV-Tax). PMA was added to the indicated reactions 30 min post-transfection, and luciferase activity was analyzed after 24 h or 48 h. Results shown in Figure 2A demonstrate that basal activation of the LTR was enhanced approximately fourfold by inducing U-937 cells to differentiate with PMA. Deletion of the TRE upstream of the TATAA box abrogated PMA- and Tax-mediated LTR activation. The effects of PMA and Tax were synergistic with respect to Tax-mediated transactivation of the HTLV-1 LTR in U-937 cells. Synergistic activation of HTLV-1 viral gene expression by phorbol esters (e.g., PMA, also known as TPA) and Tax has been demonstrated previously to occur through functionally distinct pathways [56 ].


Figure 2
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  		Figure 2. PMA-induced monocytic differentiation of U-937 cells activates HTLV-1 viral gene expression in the absence or presence of Tax. (A) U-937 promonocytic cells (1x106) were transiently transfected with a HTLV-1 LTR luciferase reporter construct (pU3R-luc; 200 ng) or a HTLV-1 minimal promoter construct (pU3R{Delta}-77-luc; 200 ng) in the absence or presence of an expression vector encoding HTLV-1 Tax (pCMV-Tax; 200 ng). PMA (500 nM) was added to the appropriate reactions 30 min post-transfection. Relative luciferase activity was quantitated 24 h post-transfection and normalized to basal pU3R-luc. (B) Cell surface markers associated with monocytic differentiation (CD11b, CD11c, CD14, CD69) were analyzed on TUR cells, a PMA-unresponsive, U-937 derivative cell line, by FACS analysis. TUR cells were left untreated (dotted lines), treated with PMA (solid lines), or treated with VD3 (shaded areas). The relative level of expression is indicated on the x-axis, and the number of events (cell counts) is indicated on the y-axis. The number represents percentage of corresponding marker-positive cells. (C) TUR cells (1x106) were transiently transfected with HTLV-1 LTR luciferase reporter constructs as described above. VD3 (500 nM) was added to the appropriate cells 30 min post-tranfection. Relative luciferase activity was quantified 48 h post-transfection and normalized to basal LTR activity. (A and C) Values shown represent mean ± SD (n=3).

 
To determine whether this result was an effect of differentiation as opposed to a PMA effect, a U-937-derivative cell line (TUR), which does not differentiate in response to PMA, was examined. TUR is a stably transfected cell line generated from U-937 cells by transfecting cells by electroporation with the pMG2neoPA plasmid containing the neomycin-resistant gene and then selected in medium containing G418 and PMA [70 ]. TUR cells retain monocytic properties but are unresponsive to phorbal ester treatment. Although human U-937 cells treated with PMA undergo monocytic differentiation, the TUR cell line does not differentiate in response to PMA [70 ]. However, it has been shown that TUR cells retain the capacity to undergo monocytic differentiation when treated with VD3 or okadaic acid, indicating that resistance of these cells to phorbal esters is related to defects in pathways activated by TPA [70 ]. Therefore, a cell-surface phenotypic analysis was performed with TUR cells in the absence or presence of PMA or VD3. The cells were analyzed for the expression of monocyte differentiation markers by flow cytometry. As expected, the percentage of TUR cells expressing CD11b, CD11c, CD14, and CD69 increased following VD3 treatment, whereas the PMA-treated TUR cells exhibited a profile similar to untreated TUR cells (Fig. 2B) .

Experimentation was then performed to determine whether VD3-induced, differentiating TUR cells could also support induction of LTR activation. Results shown in Figure 2C demonstrate that in VD3-treated cells, Tax-mediated activation of LTR activity was enhanced by ~14-fold. The absence of TRE upstream of the TATAA box abrogated VD3- and Tax-mediated LTR activation.

Enhanced transcription factor binding to TRE-1 repeat II during induction of monocytic differentiation
Sequences encoded within the HTLV-1 LTR between –242 and –117, with respect to the transcription initiation site, have been demonstrated previously to confer responsiveness to phorbol esters [56 ]. This TPA-responsive element spans sequences encoding TRE-2 (–163 to –117), TRE-1 repeat II (–183 to –163), and a portion of the TRE-1 repeat I (–251 to –231; Fig. 3 ). Differentiation agents such as PMA induce activation of the PKC pathway, resulting in activation of NF-{kappa}B and AP-1 transcription factors [40 , 46 ]. Futhermore, the activation of PKC as well as the DNA-binding activity of NF-{kappa}B and AP-1 have been shown to be up-regulated by HTLV-1 Tax [58 , 71 ]. As NF-{kappa}B and AP-1 have been shown to interact physically with the viral promoter within TRE-2 and TRE-1 repeat II, respectively [15 , 52 , 53 , 72 ], it was hypothesized that activation of one or both of these transcriptional regulators may promote up-regulation of HTLV-1 LTR activation during monocytic differentiation. To determine whether PMA-induced monocytic differentiation led to enhancement of NF-{kappa}B- and/or AP-1-specific DNA-protein complex formation, EMS analyses were performed using nuclear extracts derived from untreated or PMA-treated U-937 cells and radiolabeled probes corresponding to the TRE-1 repeats I, II, and III and TRE-2. Radiolabeled probes corresponding to the c-Myb, NF-{kappa}B, Sp1, and ATF/CREB consensus sequences were also included for comparative analysis. Results presented in Figure 4 demonstrated that PMA-induced differentiation of U-937 cells led to enhanced binding of nuclear proteins to TRE-1 repeat II. It is interesting that enhanced DNA-protein complex formation was also detected using a CREB consensus sequence, which is able to bind ATF/CREB and AP-1 family members [48 , 49 ]. Although ATF/CREB and AP-1 factors have been shown previously to interact with TRE-1 repeat II [15 , 52 , 53 ], ATF/CREB DNA-binding activity is not known to be induced by PMA [73 ]. Furthermore, ATF/CREB binding to TRE-1 repeat I and III probes was not enhanced by PMA-induced monocytic differentiation, suggesting that induction of monocytic differentiation stimulated AP-1 but not ATF/CREB binding to TRE-1 repeat II. Although DNA-protein complex formation with the NF-{kappa}B consensus sequence was enhanced in PMA-treated U-937 cells, no such enhancement was detected with TRE-2, suggesting that NF-{kappa}B was not involved in LTR activation during PMA-induced monocytic differentiation. Based on these results, it was hypothesized that PMA-induced monocytic differentiation promoted activation of HTLV-1 LTR-directed viral gene expression, primarily through triggering AP-1 DNA-binding activity to TRE-1 repeat II.


Figure 3
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  		Figure 3. v-Jun- and PMA-responsive elements of the HTLV-1 LTR include sequences spanning TRE-1 repeat II. Previous studies [56 ] have indicated that the v-Jun- and PMA-responsive elements within the HTLV-1 LTR (–242 to –117; underlined) overlap sequences that comprise TRE-1 repeat II and TRE-2.

 

Figure 4
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  		Figure 4. Enhanced binding of nuclear proteins to TRE-1 repeat II during monocytic differentiation. EMS analyses were performed by reacting nuclear extracts derived from untreated or PMA-treated U-937 cells with radiolabeled, double-stranded oligonucleotides corresponding to TRE-1 repeat I, TRE-1 repeat II, TRE-1 repeat III, TRE-2, c-Myb, NF-{kappa}B, Sp1/3, or CREB consensus sequences for 30 min at 30°C. DNA-protein complex-containing reactions were then subjected to electrophoresis through a 5% polyacrylamide gel at 200 V for ~2 h and detected by autoradiography. Open arrow indicates the DNA-protein complexes, which were induced in PMA-treated nuclear extracts. Closed arrows indicate nonspecific DNA-protein complexes.

 
AP-1 proteins bind to the TRE-1 repeat II in cells of monocytic origin
To demonstrate that the inducible DNA–protein complex formed on the TRE-1 repeat II consisted of AP-1 components, EMS supershift analyses were performed using nuclear extracts derived from untreated or PMA-treated U-937 cells, a radiolabeled probe corresponding to TRE-1 repeat II, and polyclonal antibodies directed against individual AP-1 family proteins (c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD). Induction of monocytic differentiation with PMA increased transcription factor-binding to TRE-1 repeat II, as shown in Figure 5A , from which supershifted complexes could be detected by addition of antibody directed against JunB. Unfortunately, the supershifted complexes exhibited similar mobility to that of existing DNA-protein complexes, previously shown to consist of ATF/CREB factors (previously designated as C1) [15 , 52 , 53 ]. Therefore, to alleviate this problem, additional EMS supershift analyses were performed using a mutant TRE-1 repeat II probe, MII 12,13,14(GTG:TAA), previously shown to block the formation of specific ATF/CREB DNA–protein complexes (C1 and C2) but not affect AP-1 DNA–protein complex formation [15 , 53 ]. In addition, polyclonal antibodies that recognize a conserved region within all Fos or all Jun proteins were used to facilitate the detection of AP-1-specific DNA–protein complexes. In the absence of higher molecular weight ATF/CREB complexes, the results in Figure 5B demonstrate more clearly the presence of JunB-supershifted complexes. Furthermore, additional, although minor supershifted DNA–protein complexes, could be readily detected by addition of antibodies directed against Fra-1, Fra-2, and JunD (Fig. 5B) . The contribution of AP-1 to the formation of differentiation-induced TRE-1 repeat II-specific DNA–protein complexes became most evident upon addition of polyclonal antibody directed against a conserved region present in all Jun family proteins (compare Fig. 5B , lanes 5 and 8).


Figure 5
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  		Figure 5. Differentiation-induced DNA–protein complexes formed on TRE-1 repeat II contain components of the AP-1 transcription factor family. EMS supershift analyses were performed by reacting nuclear extracts derived from untreated [antibody (Ab)] or PMA-treated U-937 cells (A and B) with radiolabeled, double-stranded oligonucleotides, corresponding to the parental (A) or mutant (B) TRE-1 repeat II for 30 min at 30°C. Control serum (CS), polyclonal antibody specific for all Fos family members (FOS), all Jun family members (JUN), or polyclonal antibody specific for individual members of the AP-1 family (c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB, and JunD) were added to the appropriate reactions during the final 15 min incubation. DNA–protein complex containing reactions were then subjected to electrophoresis through a 5% polyacrylamide gel at 200 V for ~2 h and detected by autoradiography. Open arrowheads indicate supershifted DNA–protein complexes.

 
Dominant-negative AP-1 (A-Fos) prevents differentiation-induced activation of the HTLV-1 LTR
Previous studies have shown that v-Jun stimulation of the HTLV-1 LTR was dependent on sequences located between –242 and –101 [51 ]. The observation that components of the AP-1 family of transcription factors form PMA-inducible DNA–protein complexes with TRE-1 repeat II suggested that AP-1 may play a crucial role in HTLV-1 LTR activation during monocytic differentiation. To examine directly whether up-regulation of HTLV-1 viral gene expression during monocytic differentiation was mediated by AP-1, dominant-negative AP-1 (A-Fos) and ATF/CREB (A-CREB) expression vectors were used in transient expression analyses. A-ZIP expression vectors encode proteins containing a leucine zipper dimerization domain derived from one of the members of the targeted bZIP transcription factor family (e.g., c-Fos or CREB-1) and an acidic extension [74 , 75 ]. A-ZIP proteins heterodimerize with and neutralize the DNA-binding activity of the targeted, endogenous bZIP transcription factor family. The activity and specificity of these dominant-negative inhibitors have been demonstrated previously and are mediated through the leucine zipper, a region highly conserved within bZIP families [76 ]. The effectiveness of A-Fos and A-CREB with respect to disrupting c-Jun- and CREB-1-mediated transcriptional activation was demonstrated by transient expression analyses in U-937 cells (Fig. 6A ). The results in Figure 6B demonstrate that expression of A-Fos inhibited basal LTR activation modestly in untreated U-937 cells. However, expression of A-Fos almost completely blocked basal activation of the HTLV-1 LTR in U-937 cells stimulated to differentiate with PMA. As expected, expression of A-CREB also down-regulated basal LTR activation in untreated and PMA-treated U-937 cells. It is surprising that expression of A-Fos reduced Tax-mediated transactivation of the HTLV-1 LTR, approximately twofold in uninduced and PMA-induced U-937 cells (Fig. 6B) . Not surprising is the observation that expression of A-CREB totally abrogated Tax-mediated transactivation, which is dependent on ATF/CREB factors [12 , 17 , 51 , 77 78 79 ]. Collectively, these results suggest that AP-1 is a critical mediator of basal HTLV-1 LTR activation and to a much lesser extent, Tax-mediated transactivation during PMA-induced monocytic differentiation. We have also tested the effect of AP-1 on VD3-differentiated TUR cells and observed similar results in the case of Tax-mediated LTR activation; however, basal LTR activation increased slightly in the presence of A-Fos and A-CREB (Fig. 6C) .


Figure 6
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  		Figure 6. Inhibition of AP-1 DNA-binding activity abrogates differentiation-induced HTLV-1 LTR activation. (A) U-937 cells (1x106) were transiently transfected with ATF/CREB (pCRE-luc; 200 ng) or AP-1 (pAP-1-luc; 200 ng) luciferase reporter vectors in the absence or presence of expression vectors encoding CREB-1 (pCMV-CREB-1; 500 ng), c-Jun (pCMV-c-Jun; 500 ng), dominant-negative AP-1 (A-Fos; 500 ng), and/or dominant-negative CREB (A-CREB; 500 ng). Relative luciferase activity was quantitated 24 h post-transfection and normalized to basal pCRE-luc. The values shown represent mean ± SD (n=3). (B) U-937 cells (1x106) and (C) TUR cells were transiently transfected with a HTLV-1 LTR luciferase reporter construct (pU3R-luc; 200 ng) or a HTLV-1 minimal promoter construct (pU3R{Delta}-77-luc; 200 ng) in the absence or presence of expression vectors encoding Tax (pCMV-Tax; 200 ng), dominant-negative AP-1 (A-Fos; 500 ng), or dominant-negative CREB (A-CREB; 500 ng). PMA was added to the appropriate U-937 reactions, and VD3 was added to the appropriate TUR reactions 30 min post-transfection. Relative luciferase activity was quantitated 24–48 h post-transfection and normalized to basal pU3R-luc. The values shown represent mean ± SD (n=3) and are representative of at least three independent experiments. A-Fos and A-CREB significantly (*, P<0.05, vs. negative controls) inhibited basal and Tax-mediated transactivation of the full-length HTLV-1 LTR in uninduced and PMA-induced U-937 monocytic cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that HTLV-1 viral gene expression becomes activated upon differentiation of monocytic cell lines with phorbol ester. Activation of viral gene expression was mediated primarily through the enhanced DNA-binding activity of AP-1 to TRE-1 repeat II. Although previous studies have demonstrated that the HTLV-1 LTR could be activated by v-Jun and phorbol esters, this study is the first to demonstrate a connection between AP-1 binding to the HTLV-1 LTR and differentiation-induced viral gene expression. PMA has been well characterized as an activator of PKC and has been shown to induce differentiation of cell lines of the myeloid lineage [51 ]. However, other stimuli leading to differentiation of monocytes and activation of T cells are also known to activate PKC. Therefore, it appears evident that multiple signaling events may be able to promote activation of AP-1, resulting in up-regulation of HTLV-1 viral gene expression (Fig. 7 ). Tax has also been shown to up-regulate PKC activation [71 ], providing a mechanism to explain how PMA and Tax may be able to synergistically activate HTLV-1 gene expression. Although PKC has been shown to activate NF-{kappa}B and AP-1, we have clearly demonstrated that AP-1 DNA binding to TRE-1 repeat II was enhanced during induction of differentiation and that inhibition of AP-1 DNA-binding activity abrogated differentiation-induced HTLV-1 LTR activation. It remains possible that AP-1 proteins may function to activate the HTLV-I LTR in the absence or presence of existing ATF/CREB proteins through the formation of heterdimers. This is not the first report to link monocytic differentiation and activation of AP-1 to regulation of retroviral gene expression. AP-1 has previously been shown to mediate up-regulation of visna virus gene expression in differentiating monocytes [80 ], pointing to a potentially conserved mechanism of virus reactivation in monocytes.


Figure 7
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  		Figure 7. Mechanisms of AP-1-dependent differentiation-induced HTLV-1 viral gene expression. Stimuli, which trigger monocytic differentiation, activate PKC and enhance the expression and DNA-binding activity of Fos and Jun transcription factors. In cells of the monocyte/macrophage lineage, which are infected with HTLV-1, differentiation-induced AP-1 DNA binding to TRE-1 repeat II may promote activation of the HTLV-1 LTR in the absence or presence of the viral transactivator protein Tax.

 
The nature of HTLV-1 viral gene expression during primary infection, clinical latency, and disease progression remains unresolved. The low level of viral gene expression detected in peripheral blood mononuclear cells has led many investigators to speculate that HTLV-1 establishes a latent infection in vivo [81 ]. A latent infection may permit HTLV-1 to spread within dividing lymphocytes without allowing recognition by the immune system and account for the unusually long period of clinical latency observed during HTLV-1 infection. However, the development of an aggressive cellular and humoral immune response directed against HTLV-1 antigens during progression of HAM/TSP strongly suggests that viral gene expression becomes up-regulated during disease pathogenesis. Previous reports have emphasized the critical role that APCs may play in development of highly active CD4+ and CD8+ T cell responses [37 , 82 ]. Although HTLV-1 predominantly targets CD4+ T cells in vivo, infection of macrophages, DCs, and their precursors in the peripheral blood may function to initiate or exacerbate the activated state of the immune response during HAM/TSP [9 , 13 , 83 ]. The results reported herein suggest that AP-1 activation by stimuli that promote monocytic differentiation (e.g., phorbol esters) may activate HTLV-1 LTR-directed viral gene expression as these cells differentiate into macrophages, DCs, or microglial cells. This inducible program of viral gene expression may allow resting monocytes to evade immunosurveillance, and differentiated macrophages and DCs stimulate T cell activation and inflammatory cytokine production. It is interesting that HTLV-1-encoded genes have also been shown to affect the ability of monocytes to differentiate into functional DCs [82 ]. This reciprocal relationship between monocytic differentiation and viral gene expression may also extend to the differentiation and/or activation of other cell populations, including T lymphocytes [84 ].

These studies have implications for the pathogenesis of HTLV-1-associated disorders and highlight a possible mechanism, whereby HTLV-1 may emerge from a latent state to trigger a highly aggressive cellular and humoral immune response characteristic of HTLV-1-induced, neurologic disease. Previous studies have identified that the bone marrow may act as a potentially important, latent viral reservoir in patients with HAM/TSP [83 ]. Resident bone marrow progenitor cells likely become exposed to virus through the normal trafficking of productively infected CD4+ T cells from the peripheral blood through the bone marrow vasculature. As a result, latently infected bone marrow progenitor cells may slowly accumulate in the bone marrow over time and continually seed the peripheral blood with infected lymphoid and myeloid lineage precursors, which eventually differentiate into functionally competent immune cells carrying HTLV-1 proviral DNA. Future studies will be necessary to elucidate the role of HTLV-1 infection and viral gene expression in APCs and their precursors in the pathogenesis of HAM/TSP.


   ACKNOWLEDGEMENTS
 
These studies were supported by the U.S. Public Health Service/National Institutes of Health (USPHS/NIH; CA54559 awarded to B. W.). The authors thank Dr. Fred Krebs (Drexel University College of Medicine) for his advice and Dr. Charles Vinson (NIH) for providing the A-Fos and A-CREB expression vectors.

Received December 8, 2005; revised April 17, 2006; accepted April 27, 2006.


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