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Blood, 1 February 2007, Vol. 109, No. 3, pp. 1051-1060. Prepublished online as a Blood First Edition Paper on September 21, 2006; DOI 10.1182/blood-2006-06-026781.
IMMUNOBIOLOGY Human T-cell leukemia virus type-1 Tax oncoprotein regulates G-protein signaling1 Department of Cellular and Molecular Biology, Faculty of Gembloux, Gembloux, Belgium; 2 Institut de Recherche en Biologie Humaine et Moléculaire, Université Libre de Bruxelles Campus Erasme, Brussels, Belgium; 3 Laboratoire de Virologie Moléculaire, Institut de Biologie et de Médecine Moléculaires (IBMM), Service de Chimie Biologique, Université Libre de Bruxelles, Gosselies, Belgium; 4 Department of Veterinary Biosciences and Molecular Virology Immunology and Department of Medical Genetics, Center for Retrovirus Research, Comprehensive Cancer Center and Solove Research Institute, The Ohio State University, Columbus, OH; and 5 Unité d'Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Paris, France
Human T-cell leukemia virus type-1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and neurological syndromes. HTLV-1 encodes the oncoprotein Tax-1, which modulates viral and cellular gene expression leading to T-cell transformation. Guanine nucleotide–binding proteins (G proteins) and G protein–coupled receptors (GPCRs) constitute the largest family of membrane proteins known and are involved in the regulation of most biological functions. Here, we report an interaction between HTLV-1 Tax oncoprotein and the G-protein ß subunit. Interestingly, though the G-protein ß subunit inhibits Tax-mediated viral transcription, Tax-1 perturbs G-protein ß subcellular localization. Functional evidence for these observations was obtained using conditional Tax-1–expressing transformed T-lymphocytes, where Tax expression correlated with activation of the SDF-1/CXCR4 axis. Our data indicated that HTLV-1 developed a strategy based on the activation of the SDF-1/CXCR4 axis in the infected cell; this could have tremendous implications for new therapeutic strategies.
Human T-cell leukemia virus type-1 (HTLV-1), the first pathogenic retrovirus discovered in humans 26 years ago,1 is the causative agent of 2 major diseases: a rapidly fatal leukemia designated adult T-cell leukemia (ATL)2 and a neurological degenerative disease known as tropical spastic paraparesis (TSP) or HTLV-1–associated myelopathy (HAM).3 Malignancy develops in approximately 1 in 20 HTLV-1–infected persons after 40 to 50 years of latency.4 The viral transcriptional activator and oncoprotein Tax-1 has been the major focus of scientific investigation because of its numerous and crucial roles in the pathogenesis of HTLV-1–induced diseases (for reviews, see Jeang et al,5 Grassmann et al,6 and Azran et al7). The primary role of Tax-1 in the viral life cycle of HTLV-1 is to directly promote viral mRNA synthesis.8 Tax-1 acts through highly conserved 21-bp repeat elements, called Tax-1–responsive elements (TREs), located within the 5' LTR.9 Tax-1 does not bind DNA directly; rather, it acts through cellular transcription factors, such as cyclic adenosine monophosphate (cAMP) response element-binding (CREB), nuclear factor-  B (NF-B), serum responsive factor (SRF), and activator protein 1 (AP-1) transcription factors (for reviews, see Jeang et al,5 Grassmann et al,6 and Azran et al7). Tax-1 modulates the expression of an array of cellular genes directly involved in T-cell proliferation, such as interleukin-2 (IL-2) and the  subunit of its receptor (IL-2R),10,11 IL-15 and its receptor (IL-15R)12,13 granulocyte macrophage–colony-stimulating factor (GM-CSF)14 and tumor necrosis factor- (TNF-).15 Tax-1 also is involved in cell-cycle regulation by direct activation of cyclins D2 and D3 and cyclin kinases CDK4 and CDK6,16–19 by inactivating the cyclin-dependent kinase inhibitor p16,INK4A 20 or by interacting with the human mitotic checkpoint protein HsMAD1.21 HTLV-1 Tax-binding factors also include MEKK1,22 the I-B kinase,23 or the PCAF protein.24 In fact, protein-protein interactions with cellular factors are crucial for Tax-1 to perturb the regulation of many cellular pathways (for reviews, see Jeang et al,5 Grassmann et al,6 and Azran et al7).
The guanine nucleotide (GTP)–binding protein (G-protein) signal transduction network, one of the major information transfer systems, allows the cell to communicate with its surroundings and to participate in a multicellular organization. The minimum components of this system are a 7-transmembrane G-protein–coupled receptor (GPCR), a heterotrimeric complex of G-protein In this study, we report a functional interplay between HTLV-1 Tax oncoprotein and the G-protein signaling pathways. We found that the Gß subunit is a specific partner of HTLV-1 Tax and that it negatively regulates its transactivation activity over the HTLV-1 viral promoter. Conversely, we also showed that Tax-1 can activate the stromal cell–derived factor (SDF)–1/CXC chemokine receptor 4 (CXCR4) ligand/receptor axis in T-lymphocytes.
Cell culture Jurkat and MT4 cells were cultured in RPMI 1640 medium (Sigma, St Louis, MO) supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics. The same medium containing 20% fetal calf serum and 40 U/mL recombinant IL-2 was used for the propagation of Tax-transformed T-lymphocytes (Tesi), as previously described.36,37 Suppression of Tax expression in Tesi cells was achieved after a 7-day cultivation period in the presence of 1 µg/mL tetracycline. HeLa and HEK293T cells were cultured in DMEM, as previously described.38 Plasmids
Plasmids pcDNAFlag-Gß1, pcDNAFlag-Gß2, pcDNAFlag-Gß5, pcDNAG GST pull-down assay
The HB101 strain of Escherichia coli was transformed with plasmid pGexTax1, pGexTax2, pGexG Coimmunoprecipitation
HEK 293T cells were divided and seeded in 10-cm plates at a density of 2 x 106 cells/plate and were transfected the next day with 20 µg plasmid DNA through the calcium phosphate method. Western blotting and immunoprecipitation assays were performed as previously described.38,39 Briefly, cell lysates were immunoprecipitated with anti-Flag (mouse M2; Sigma) or anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA) agarose-conjugated antibodies or control antibodies. The immunoprecipitates were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using anti-Tax antibody ( Intracellular cascade assays For MAP kinase activation assay, Tesi cells, Tesi-Tax control cells (Tesi cells cultivated for 10 days in the presence of 1 µg/mL tetracycline for Tax suppression), and Tesi-PTX cells (Tesi cells cultivated for 24 hours in the presence of 100 ng/mL pertussis toxin) were stimulated for 5 minutes with 30 ng/mL SDF-1, 30 ng/mL MCP-1, or 50 ng/mL RANTES. Cells were lysed, and ERK1/2 activation was measured by Western blotting using an anti–phospho-p42/44 monoclonal antibody (E10; Cell Signaling Technology, Beverly, MA). For calcium mobilization, Tesi and Tesi-Tax control cells (107 cells/mL in HBSS without phenol red but containing 0.1% BSA) were loaded with 5 µM FURA-2 (Invitrogen, Carlsbad, CA) for 30 minutes at 37°C in the dark. The loaded cells were washed twice, resuspended at 106 cells/mL, and kept for 30 minutes at 4°C in the dark. Ca2+ mobilization in response to 50 nM SDF-1 was measured with the use of a luminescence spectrometer (LS50B; Perkin Elmer, Wellesley, MA) by recording the ratio of fluorescence emitted at 510 nm after sequential excitation at 340 and 380 nm. Binding and migration assays Experiments were performed to measure the binding of SDF-1 to membranes from Tesi cells. Samples containing 5 µg membrane proteins, prepared as described,42 were combined with 10 nM 125I-SDF-1 in 100 µL final volume of assay buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA), followed by incubation for 90 minutes at 25°C. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled SDF-1. Bound SDF-1 was separated by filtration through GF/B filters presoaked in 0.5% polyethylenimine (Sigma-Aldrich, Poole, United Kingdom). Filters were counted in a ß scintillation counter. For T-cell migration assays, Transwell culture chambers (5-µm pore size; Costar, Cambridge, MA) were used. The lower wells were filled with 500 µL medium (RPMI 1640 containing 1% FCS) containing 30 ng/mL SDF-1, 50 ng/mL RANTES, or 30 ng/mL MCP-1. Tesi cells (105 cells) suspended in 100 µL medium were loaded into the upper wells. After incubation for 2 hours at 37°C, the cells that had migrated to the lower wells were counted and shown as percentages of the input cells. Flow cytometry For quantification of CXCR4 receptor expression on the cell surface, Tesi cells were incubated with a polyclonal rabbit anti-CXCR4 antibody (Calbiochem, San Diego, CA) followed by a PE-conjugated antirabbit secondary antibody. Flow cytometry analyses were performed with a FACScan (BD Biosciences, San Jose, CA). Confocal microscopy
Two micrograms plasmids (CMVTax, pTax2, pSGTax, pcDNAFlag-Gß1, pcDNAFlag-Gß2, and pcDNAFlag-Gß5) were transfected into HeLa cells with the use of Genejammer (Stratagene, La Jolla, CA). Twenty-four hours after transfection, cells were fixed in 3.7% formaldehyde (20 minutes at 4°C), permeabilized with 0.1% Nonidet P40 (10 minutes), and incubated with anti-Flag antibodies (Sigma) or anti-Tax antibodies ( Luciferase assays
Ten micrograms reporter plasmids pLTR1-Luc, p
Figure S1 (available on the Blood website; see the Supplemental Figures link at the top of the online article) shows pull-down of the Gß but not the G
Gß interacts with Tax oncoproteins To gain insight into the molecular mechanisms that govern Tax functions, we performed yeast 2-hybrid screening and identified several Tax interactors.39 Two of these clones appeared to be related to the human Gß2 coding sequence (accession no. XP005013). To confirm the binding specificity of Tax-1 to Gß2, HEK293T cells were transfected with expression constructs for Tax-1 and Flag-tagged Gß2. After cell lysis, Flag-Gß2 was immunoprecipitated with an anti-Flag antibody. Immunoprecipitates then were resolved by SDS-PAGE and analyzed by Western blotting with an anti–Tax-1 antibody. As shown in Figure 1A, Tax-1 specifically copurified with Flag-Gß2 when both proteins were expressed together. As control, no Tax-1 was detected in immunoprecipitates from cells expressing either Tax-1 or Flag-Gß2 alone (Figure 1A, IP).
To generalize our findings, we then tested 2 other Gß subunits, Gß1 and Gß5, for their ability to interact with Tax-1. As with Gß2, coimmunoprecipitation experiments revealed a robust interaction of Tax-1 with Gß5 and, to a lesser extent, with Gß1 (Figure 1B). We next analyzed the ability of endogenous Gß proteins to interact with Tax in an HTLV-1–transformed cell line (MT4) and a Tax-transformed T-cell line (Tesi). To this end, MT4, Tesi, or Jurkat cells were lysed, and Gß proteins were immunoprecipitated with an anti-Gß antibody. Immunoprecipitates were then analyzed by Western blot with an anti–Tax-1 antibody. As shown on Figure 1C, Tax-1 coimmunoprecipitated with Gß proteins in MT4 and Tesi cells. We also used GST–Tax-1 fusion proteins bound to Sepharose beads to precipitate endogenous Gß subunits from HeLa and Jurkat cells. As shown on Figure 1D, Gß proteins from HeLa or Jurkat cells coprecipitated with GST–Tax-1 but not with GST alone. These data demonstrated that physiological levels of Gß subunits also interacted with HTLV-1 Tax.
G-protein complexes, made up of Gß We next tested whether Gß could associate with Tax-2B protein from HTLV-2. To this end, we used the GST pull-down assay and found that significant amounts of the in vitro–translated Gß2 protein were pulled down by GST–TaxHTLV-1 and GST–TaxHTLV-2 fusion proteins compared with the GST control (Figure 1E). In addition, the Tax protein from bovine leukemia virus (BLV) also was associated with Flag-tagged Gß2 in 293T cell lysates (Figure 1F). These data strongly suggested that binding to Gß is a general property of deltaretrovirus-encoded Tax oncoproteins. Tax-1 interacts with Gß through its WD repeat domains
The crystal structure of Gß revealed that the protein is made of 2 distinct regions: an amino-terminal
Tax-1 has 2 sites of interaction with Gß To map the Tax-1 domains involved in Gß-binding, HEK293T cells were cotransfected with Flag-Gß2 and full-length GFP-tagged Tax-1 (GFP-Tax-1) or 4 different GFP-tagged Tax-1–deletion mutants (Figure 2C).45 Flag-Gß2 was immunoprecipitated from transfected cell lysates, and coimmunoprecipitated GFP–Tax-1 proteins were revealed by immunoblotting. As shown in Figure 2D, the deletion mutants TD99 and TD254 associated with Gß2, whereas the mutants TD55 and TD150 did not show any detectable binding. These results indicated that 2 distinct regions of Tax-1, amino acids 55-92 and a central domain (aa 150-198), are involved in Gß-binding. Gß subunits colocalize with HTLV-1 Tax
The heterotrimeric G-protein complexes, through a prenylated cysteine residue on its G To examine the subcellular localization of Gß and Tax-1 proteins, HeLa cells were transfected with constructs expressing Tax-1 or Flag-tagged versions of Gß1 and Gß2. The cells then were stained with an anti-Flag antibody followed by Alexa 546–conjugated secondary antibody (for Gß subunits) or an anti–Tax-1 antibody followed by a fluorescein-conjugated secondary antibody. As expected, most Gß subunit proteins were localized to the cytoplasm, with small fractions also detected in the nucleus (stained by TOPRO-3) and at the plasma membrane (Figure 3A). Tax predominantly localized to the nucleus, but substantial amounts of the protein also were found in the cytoplasm (Figure 3B). In addition, Tax-1 displayed a speckled pattern known as Tax-speckled structures (TSSs), which have been described as Tax-1 transcriptional regulation hot spots.52
We next assessed the localization of coexpressed Gß and Tax-1 proteins by transfecting HeLa cells with equal amounts of Flag-Gß and Tax-1 expression constructs (1 µg each vector) (Figures 3C, S2). In the presence of Gß2, the subcellular localization of Tax-1 was identical to that seen with Tax-1 alone (compare Tax+FITC staining in Figure 3B and Tax+FITC staining in Figure 3C, Tax-1+Gß2). Interestingly, in the presence of Gß1, Tax-1 was localized exclusively to the cytoplasm (Figure 3C, Tax+FITC staining, Tax-1+Gß1). Similar colocalization of Tax-1 was observed with Gß5 (Figure S2; Tax-1+Gß5). These experiments revealed that coexpression of Tax-1 dramatically altered the subcellular localization of Gß proteins. In the presence of Tax-1, individual Gß subunits were targeted to the TSS and completely lost their diffuse distribution in the cytoplasm and the plasma membrane (compare Figure 3C, Flag+Alexa 546 staining with Figure 3A, Flag+Alexa 546 staining). In addition, the Gß subunits also colocalized with HTLV-2 and BLV Tax oncoproteins (Figure S3 and data not shown). We concluded that coexpression of Tax proteins and Gß subunits changed their respective subcellular localization, suggesting that their association might have affected their individual functions. Gß regulates the Tax-1 transactivation of the HTLV-1 LTR promoter
The transforming potential of HTLV-1 Tax has been attributed mainly to its ability to activate gene expression through the CREB/ATF and NF-
Tax-1 expression induces CXCR4 receptor activation
In vivo, heterotrimeric G proteins transduce an extracellular signal through GPCR. Activated GPCR facilitates the exchange of GDP for GTP in the G We used immortalized T-lymphocytes that conditionally expressed Tax-1 under the control of the tetracycline operon (Tesi cells).36,37 The Tesi-Tax control cells were generated by suppressing Tax-1 expression in Tesi cells with a 7-day period of propagation in the presence of tetracycline.36,37 Immunoblot analysis with an anti–Tax-1 monoclonal antibody demonstrated the suppression of Tax-1 protein expression in Tesi-Tax control cells (Figure 5A, bottom; compare Tesi and Tesi-Tax control lanes). Tesi cells then were treated with stromal cell-derived factor-1 (SDF-1; 30 ng/mL), which specifically binds to the CXCR4 receptor; monocyte chemoattractant protein-1 (MCP-1; 30 ng/mL), a ligand for CCR1 and CCR2 receptors; or regulated on activation normal T-cell expressed and secreted (RANTES; 50 ng/mL), which binds to CCR1, CCR3, and CCR5. Cells were evaluated for cellular activation. Given that these latter chemokines have been shown to activate the extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein (MAP) kinase pathway, cell lysates were tested for the phosphorylation of ERK1/2 using a phosphospecific anti-p42/44 antibody. As shown in Figure 5A (top), stimulation of Tesi cells by SDF-1 resulted in the phosphorylation of ERK1/2 (Figure 5A; Tesi, SDF-1). The SDF-1 activation of ERK1/2 was both Tax-1 and Gi dependent because Tesi-Tax control cells, which do not express Tax-1, or Tesi-PTX cells, which had been treated with pertussis toxin for Gi inactivation, were not stimulated by SDF-1 (Figure 5A; SDF-1, Tesi-Tax control and Tesi-PTX). In contrast, MCP-1 did not activate chemokine receptors on Tesi cells (Figure 5A; MCP-1), whereas the phosphorylation of ERK1/2 after treatment of Tesi cells with RANTES was not Tax-1 dependent (Figure 5A, right; RANTES).
To confirm the role of Tax-1 in the activation of the SDF-1/CXCR4 axis, we analyzed calcium signaling in Tesi compared with Tesi-Tax control cells. To this end, cells were loaded with FURA-2, and calcium mobilization in response to SDF-1 was measured. As shown in Figure 5B, calcium signaling was activated by SDF-1 in Tesi but not in Tesi-Tax control cells, confirming the role of Tax-1 in the regulation of SDF-1/CXCR4 intracellular signaling. Chemotaxis of Tax-1–transformed T cells in response to SDF-1 also was examined using an in vitro migration assay. As shown in Figure 5C, migration of Tax-1–expressing Tesi cells toward SDF-1 was 12-fold more efficient than that of Tesi-Tax control cells. In addition, specific intracellular cascade activation and migration toward SDF-1 were confirmed with the MT4 HTLV-1–transformed cell line (Figures S4E-F). Tax-1 regulates the expression of several cellular genes involved in cellular activation, proliferation, and transformation. Furthermore, Tax-1 has been shown to be a relatively weak activator of the CXCR4 promoter.56 Therefore, we analyzed the surface expression of CXCR4 on Tesi and Tesi-Tax control lymphocytes by flow cytometry. Results presented in Figure 6A showed that surface CXCR4 receptor expression was similar between the Tesi and Tesi-Tax control cells. This demonstrated that the differences in intracellular cascade activation between Tesi and Tesi-Tax control cells were not caused by altered expression of the CXCR4 receptor.
Finally, we tested whether Tax-1 could affect the binding properties of the CXCR4 receptor. For this purpose, we compared the binding efficiency of 125I-SDF-1 to Tesi compared with Tesi-Tax control cell membrane proteins. Greater amounts of radiolabeled 125I-SDF-1 were bound specifically to Tesi membranes, suggesting that Tax-1 directly favors the SDF-1/CXCR4 interaction (Figure 6B). Together, these data indicate that Tax-1 expression in immortalized T-cell lines specifically regulates the activation of the SDF-1/CXCR4 ligand/receptor axis.
In this study, we characterized a functional interaction between the HTLV-1 Tax oncoprotein and the G-protein signaling pathway. The deltaretroviridae subgroup of retroviruses comprises bovine leukemia virus (BLV) and 3 types of primate T-cell leukemia viruses (PTLVs) namely, HTLV-1/STLV-1, HTLV-2/STLV-2, and HTLV-3/STLV-3.57,58 These viruses share similar genomic organization, and their tax genes seem to play a central role in the induced diseases. Indeed, studies using transgenic mice with Tax-1 expression restricted to developing thymocytes recently demonstrated that the TAX1 gene alone is able to induce leukemia and lymphoma identical to ATLL in humans.59 The transformation capacity of Tax-1 likely results from its interaction with numerous cellular proteins and the regulation of several cellular signaling and transcriptional control pathways.
We previously identified the Gß2 subunit using a yeast 2-hybrid screen for Tax interactors.39 In this study, we used coimmunoprecipitation and GST pull-down assays to confirm the interactions and to extend our observations to HTLV-2 and BLV Tax oncoproteins (Figure 1). It appears that HTLV-1 and -2 and BLV Tax oncoproteins interact with the Gß2 subunit. This observation suggests that a common mechanism is used by deltaretroviruses to subvert G-protein signaling. Furthermore, we showed that the Gß subunit targets Tax-1 on the same domain (aa 55-92; Figure 2) required for p300/CBP-binding60 and suppresses the transcriptional activity of the Tax-1 protein on the HTLV-1 LTR promoter without affecting its NF-
Five known human Gß subunit genes25,61 may form potential combinations with at least 12 G
Consistent with its transcriptional transactivation role, the Tax-1 protein localizes predominantly to the nucleus and contains a nuclear localization sequence (NLS) within its first 58-amino terminus.67 The cytoplasmic localization of Tax-1 is promoted by a nuclear export signal (NES) located between amino acids 188 and 200.68 With respect to its multiple nuclear and cytoplasmic functions, Tax-1 may shuttle constantly between the nucleus and the cytoplasm.69 The Gß
Many viruses have developed strategies to hijack the cellular G-protein signaling transduction network. DNA viruses such as herpesviruses encode chemokine GPCRs critical for viral replication and viral-induced diseases in natural hosts.73 Among retroviruses, human immunodeficiency virus (HIV) uses 2 cellular host GPCRs, CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4), as coreceptors for cell entry.74 Little is known about the role of G proteins and GPCRs in the HTLV-1 life cycle and pathogenesis. However, it has been shown that CCR4 and CCR7 frequently are expressed in ATL cells and may contribute to tissue infiltration by circulating leukemic cells and HTLV-1–infected T cells.75–77 In addition, chemokines such as MCP-1, RANTES, MIP-1– The Tax1 protein is known as a potent transactivator that regulates the expression of several genes, and it was demonstrated previously that Tax-1 is able to activate the CXCR4 promoter after stimulation by PMA/ionomycin.56 Our study demonstrated that Tax-1 expression in primary T lymphocytes did not result in altered cell surface expression of CXCR4 (Figure 6A) but specifically increased the response to SDF-1 as measured by calcium mobilization, MAPK activation, or cell chemotaxis. In agreement with our functional data, we showed that the binding of radiolabeled SDF-1 was increased in membrane preparations from T cells expressing Tax-1 protein (Figure 6B). These results suggest that Tax-1 could modify the binding properties of the CXCR4 chemokine receptor.
Recently, it was reported that heterotrimeric G proteins are precoupled to GPCRs in the absence of agonist.82 It also is generally accepted that the active state of some GPCRs is stabilized by their association with a guanine nucleotide-free G-protein
In conclusion, we provided molecular evidence showing a functional interaction between Tax oncoproteins and G-protein signaling. We propose a model in which, after T-cell infection by HTLV-1, Tax-1 would induce the expression of several proteins, including cytokines and chemokines. Tax-1 also would migrate to the cytoplasm and the plasma membrane, bind to the Gß
Contribution: J.C.T. and J.-Y.S. designed and performed the research and analyzed the data. M.B., J.-F.D., J.D., and P.U. provided technical help on experiments presented in Figures 1 to 5. A.B. contributed critical reading of the manuscript. L.W. provided key reagents and contributed to research presented in Figure 3. F.D., D.P., C.V.L., P.L.G., R.M., and M.P. provided vital new reagents (antibodies, cell lines, and DNA constructions). J.-C.T., F.D., and R.K. wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Jean-Claude Twizere, Cellular and Molecular Biology, Faculty of Gembloux, 13 avenue Maréchal Juin, B-5030 Gembloux, Belgium; e-mail: twizere.jc{at}fsagx.ac.be.
This work was supported by the Belgian program on Interuniversity Poles of Attraction, Prime Minister's Office, Science Policy Programming (R.K., M.P.), the Belgian Foundation against Cancer (R.K.), the Fonds National de la Recherche Scientifique (J.C.T., M.B., P.U., F.D., C.V.L., L.W., R.K.), the National Institutes of Health (grant CA100730) (P.L.G.), and the INSERM (R.M.). We thank Dr S. Herlitze (Case Western Reserve University, Cleveland, OH), Dr B. Denker (Harvard Institute of Medicine, Boston, MA), Dr F. Bex (Free University of Brussels, Belgium), Dr H. Hamm (Department of Pharmacology, Vanderbilt University, Nashville, TN), Dr N. Gautam (Department of Anesthesiology, Washington University School of Medicine, St Louis, MO), Euroscreen S.A (Gosselies, Belgium), Dr R. Grassmann (Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg, Erlangen, Germany), Drs. K.T. Jeang and J-M. Peloponese (Molecular Virology Section, Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, MD), the National Institutes of Health AIDS Research and Reference Reagent Program (Germantown, MD), and the University of Missouri cDNA Resource Center (University of Missouri, Rolla, MO) for providing reagents.
Submitted June 1, 2006; accepted September 18, 2006.
Prepublished online as Blood First Edition Paper, September 21, 2006
DOI: 10.1182/blood-2006-06-026781
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
subunits, and an intracellular effector molecule (for a review, see Clapham and Neer
