COMMUNICATION
IKKgamma Mediates the Interaction of Cellular Ikappa B Kinases with the Tax Transforming Protein of Human T Cell Leukemia Virus Type 1*

Zhi-Liang ChuDagger §, Young-Ah ShinDagger , Jin-Ming YangDagger , Joseph A. DiDonato, and Dean W. BallardDagger §parallel

From the Dagger  Howard Hughes Medical Institute and the § Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295 and the  Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The Tax oncoprotein of human T cell leukemia virus type 1 constitutively activates transcription factor NF-kappa B by a mechanism involving Tax-induced phosphorylation of Ikappa Balpha , a labile cytoplasmic inhibitor of NF-kappa B. To trigger this signaling cascade, Tax associates stably with and persistently activates a cellular Ikappa B kinase (IKK) containing both catalytic (IKKalpha and IKKbeta ) and noncatalytic (IKKgamma ) subunits. We now demonstrate that IKKgamma enables Tax to dock with the IKKbeta catalytic subunit, resulting in chronic Ikappa B kinase activation. Mutations in either IKKgamma or Tax that prevent formation of these higher order Tax·IKK complexes also interfere with the ability of Tax to induce IKKbeta catalytic function in vivo. Deletion mapping studies indicate that amino acids 1-100 of IKKgamma are required for this Tax targeting function. Together, these findings identify IKKgamma as an adaptor protein that directs the stable formation of pathologic Tax·IKK complexes in virally infected T cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

During an adaptive immune response, antigen-stimulated CD4+ T lymphocytes become committed to an activation program that triggers a transient phase of clonal expansion (1). In contrast, infection with human T cell leukemia virus type 1 (HTLV-1)1 can lead to the loss of cell cycle control and development of an aggressive malignancy called adult T cell leukemia (2). The Tax oncoprotein encoded by HTLV-1 stimulates the constitutive nuclear expression of transcription factor NF-kappa B, which regulates antigen-directed T cell proliferation (3, 4). Studies with Tax-transgenic mice suggest that this viral/host interaction is required to maintain the transformed phenotype of HTLV-1-infected cells (5).

In quiescent T cells, the activity of NF-kappa B is controlled from the cytoplasmic compartment by virtue of its signal-dependent interaction with inhibitors, including Ikappa Balpha (6). Recent studies have identified two cytokine-inducible Ikappa B kinases (IKKs), termed IKKalpha and IKKbeta , that target Ikappa Balpha for degradation via phosphorylation at Ser-32 and Ser-36 (7). These two kinases form heterodimers and function as catalytic subunits within a 700-900-kDa multicomponent complex (8). Whereas IKKalpha and IKKbeta are activated transiently in cells treated with the cytokine tumor necrosis factor-alpha (TNF) (8-10), Tax induces their constitutive expression in HTLV-1-infected T cells (11, 12). We have recently found that Tax-induced activation of both IKK and NF-kappa B requires the formation of Tax·IKK complexes (12). However, the precise mechanism of Tax action on IKKs remains unclear.

Here we provide several lines of experimental evidence indicating that Tax-directed IKK activation is mediated by IKKgamma (also called NEMO, IKKAP1, or FIP-3), a recently identified subunit of TNF-responsive IKKs whose precise signaling function is unknown (13-16). First, interference with IKKgamma expression in T cell transfectants inhibits Tax-mediated activation of NF-kappa B. Second, IKKgamma and Tax interact stably in the context of a high molecular mass Ikappa B kinase derived from HTLV-1-infected T cells. Third, overexpression of IKKgamma in vivo is sufficient to target Tax specifically to ectopic IKKbeta , whereas deletion of the N-terminal region of IKKgamma eliminates this targeting function. The finding that IKKgamma enables Tax to dock with cellular Ikappa B kinases highlights an important missing link in the mechanism by which this oncoprotein activates the constitutive expression of NF-kappa B in HTLV-1-infected T cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Reagents-- Rabbit antisera specific for IKKgamma and Tax (amino acids 321-353) have been described (13, 17). Anti-IKKalpha (H-744) and IKKbeta (H-470) antibodies were obtained from Santa Cruz, Inc. Agarose beads conjugated to monoclonal anti-FLAG and anti-Myc antibodies were purchased from IBI-Kodak and Santa Cruz, respectively. Expression vectors for antisense IKKgamma RNA (AS-IKKgamma ) (14), Tax (17), FLAG epitope-tagged IKKbeta (10), and IKKgamma (13) have been described. Deletion mutants of IKKgamma were constructed by polymerase chain reaction using specific oligonucleotide primers (sequences available upon request) and subcloned into pcDNA3.1/Myc-His (Invitrogen). Chloramphenicol acetyltransferase (CAT) reporter plasmids contained either two tandem kappa B enhancers (kappa B-TATA-CAT) (18) or the HTLV1 5' long terminal repeat (HTLV1 LTR-CAT) (19).

Cell Culture, Transfections, and CAT Assays-- Jurkat T cells, RIP-deficient Jurkat T cells (20), HTLV-1-infected T cells (21, 22), and S107 plasmacytoma cells (23) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics. Jurkat and S107 cells were transfected via electroporation (24). Human 293T cells (25) were transfected using calcium phosphate precipitation (26). All CAT assays were performed as described (24).

Subcellular Fractionation and Biochemical Analyses-- Cytoplasmic extracts were prepared by detergent lysis (27) in the presence of phosphatase and protease inhibitors (12). For gel filtration (9), cytosolic proteins (10 mg) were equilibrated in ELB buffer (24) and subjected to chromatography on a precalibrated Superose 6 column (Amersham Pharmacia Biotech). Unless indicated otherwise, immunoprecipitations were performed as described (12). Resultant immunocomplexes were fractionated by SDS-polyacrylamide gel electrophoresis and probed on polyvinylidine difluoride membranes using an enhanced chemiluminescence system (SuperSignal, Pierce). Ikappa B kinase activity was measured as described using recombinant glutathione S-transferase protein fused to amino acids 1-54 of Ikappa Balpha (GST-Ikappa Balpha ) (9, 12).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Constitutive Activation of NF-kappa B by Tax Involves IKKgamma but Not RIP-- In prior studies, we established that Tax binds to and persistently activates a TNF-responsive Ikappa B kinase containing two catalytic subunits termed IKKalpha and IKKbeta (12). To determine how Tax interfaces with cellular IKKs, we first examined whether the Tax·IKK signaling axis involves the death domain kinase RIP, which is essential for TNF-induced activation of NF-kappa B (20, 28). For these studies, Jurkat human T cells containing a RIP null mutation (20) were cotransfected with a Tax expression vector (Tax-WT) and a CAT reporter plasmid containing two kappa B enhancers (kappa B-TATA-CAT). Parallel experiments were conducted with expression vectors containing point mutations that selectively disrupt the ability of Tax to access either the CREB/ATF (Tax-M47) or the NF-kappa B/Rel (Tax-M22) transcription factor pathway (17). As shown in Fig. 1A, Tax-WT potently stimulated NF-kappa B-directed transcription in both parental and RIP-deficient Jurkat T cells. Similar results were obtained with Tax-M47 but not Tax-M22, consistent with their differing capacities to activate TNF-responsive IKKs (12). These in vivo functional data clearly show that RIP is dispensable for Tax-induced activation of NF-kappa B.


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Fig. 1.   IKKgamma is required for Tax-induced activation of NF-kappa B in human T cells. A, wild type or RIP-deficient (20) Jurkat T cells (1 × 107) were cotransfected with kappa B-TATA-CAT (2.5 µg) and the indicated Tax expression vector (5 µg). Whole cell extracts were prepared after 48 h and assayed for CAT activity. Results from triplicate transfections are reported as the mean fold induction (± S.E.) of CAT activity over basal levels in cells transfected with kappa B-TATA-CAT alone. B, Jurkat cells were cotransfected with kappa B-TATA-CAT or HTLV1 LTR-CAT (2.5 µg), a Tax expression plasmid (5 µg), and the indicated doses of an antisense IKKgamma vector (AS-IKKgamma ). Whole cell extracts were prepared after 48 h and assayed for CAT activity. For each titration point (n = 3), results are expressed as the mean percentage of CAT activity relative to that in AS-IKKgamma -deficient cells (58- and 81-fold induction for kappa B-TATA-CAT and HTLV1 LTR-CAT, respectively), which was normalized to 100%.

RIP interacts specifically with IKKgamma , an integral subunit of TNF-responsive IKKs (13-16). In this regard, Yamaoka et al. (13) have reported experiments with IKKgamma -deficient rat fibroblasts, suggesting a requirement for this subunit in coupling Tax to NF-kappa B, whereas others (29) have identified an IKKgamma -deficient pre-B cell line that is fully responsive to Tax. To determine whether IKKgamma couples Tax to NF-kappa B in a more physiologically relevant setting, Jurkat T cells were cotransfected with Tax-WT, kappa B-TATA-CAT, and graded amounts of a vector that directs the synthesis of antisense IKKgamma RNA (AS-IKKgamma ). As shown in Fig. 1B, AS-IKKgamma inhibited Tax-induced transcription directed from the NF-kappa B-responsive reporter in a dose-dependent fashion. In contrast, interference with IKKgamma protein expression failed to affect Tax-induced transcription from the HTLV-1 5' long terminal repeat, which is activated by an NF-kappa B-independent mechanism (30). These functional studies demonstrate that IKKgamma is required for the induction of NF-kappa B by Tax in the context of human T lymphocytes, the in vivo target for HTLV-1.

Tax Interacts Stably with TNF-responsive IKKs Containing IKKgamma -- IKKgamma assembles with TNF-responsive IKKs primarily via its interaction with IKKbeta (13-15). This catalytic subunit also associates with Tax in HTLV-1-infected T cells (12). To determine whether IKKgamma is a core component of Tax-responsive IKKs, we first performed in vitro kinase assays using cytoplasmic extracts from SLB-1 and C8166 T cells. Whereas SLB-1 cells produce replication-competent virions (21), C8166 cells harbor a defective provirus that selectively expresses Tax (22). In these biochemical experiments, Tax, IKKalpha , IKKbeta , and IKKgamma were isolated by immunoprecipitation and assayed for IKK catalytic activity using a GST-Ikappa Balpha fusion protein as substrate. As shown in Fig. 2A (lanes 2-4, top panel), GST-Ikappa Balpha phosphorylating activity was readily detected in Tax and IKKalpha ·IKKbeta immunoprecipitates derived from SLB-1 cells. A significant amount of IKK activity was also detected in IKKgamma immunoprecipitates (lane 5). Similar results were obtained with C8166 cells (Fig. 2A, bottom panel). This subunit compositional analysis establishes that IKKgamma is associated with a constitutively active Ikappa B kinase in both HTLV-1-infected and Tax-expressing T cells.


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Fig. 2.   IKKgamma associates stably with Tax-activated IKKs in HTLV-1-infected T cells. A, subunit composition of constitutively active IKKs. Cytoplasmic extracts (200 µg) from SLB-1 and C8166 cells were subjected to immunoprecipitation with the indicated antisera. Resultant immune complexes were incubated with GST-Ikappa Balpha in the presence of [gamma -32P]ATP. Phosphoproteins were resolved by electrophoresis and visualized by autoradiography. B, size distribution of Tax-associated IKKs. Cytosolic protein extracts (10 mg) from SLB-1 cells were fractionated by gel filtration on Superose 6, and the resultant eluates were subjected to immunoprecipitation with anti-Tax antibodies. Immunoprecipitates were either analyzed for Ikappa B kinase activity as described above (top panel) or probed on immunoblots with anti-Tax antibodies (bottom panel). The horizontal bar denotes the elution position of peak IKK kinase activity derived from TNF-treated Jurkat cells. C, IKKgamma associates with Tax·IKK complexes. Pooled fractions corresponding to peak IKK activity (panel B) were subjected to immunoprecipitation with the indicated antisera, and the resultant immune complexes were analyzed for either IKK kinase activity (top panel) or Tax protein on immunoblots (bottom panel). D, stability of IKKgamma ·Tax complexes. Pooled fractions corresponding to peak IKK activity (panel B) were subjected to immunoprecipitation with IKKgamma -specific antibodies. Resultant immunocomplexes were washed three times with ELB buffer (24) containing the indicated concentration (Conc.) of dissociation agents and then assayed for either IKK kinase activity (top panel) or Tax protein (bottom panel) as described above.

The TNF-responsive form of IKK that contains IKKgamma corresponds to a 700-900-kDa multisubunit complex (13, 14). To explore the size distribution of Tax-associated IKKs, cytosolic proteins from HTLV-1-infected SLB-1 cells were fractionated by gel filtration and the resultant eluates were subjected to immunoprecipitation with anti-Tax antibodies. Consistent with the size of TNF-responsive IKKs, the majority of constitutively active IKKs associated with Tax in SLB-1 cells were detected in fractions corresponding to a molecular mass exceeding 700 kDa (Fig. 2B). To determine whether IKKgamma was also present in these Tax·IKK complexes, SLB-1 fractions containing peak kinase activity were subjected to immunoprecipitation with IKKgamma -specific antibodies and assayed for the presence of either IKK activity or Tax protein. As shown in Fig. 2C, Ikappa B kinase activity and Tax were readily detected in these IKKgamma immunoprecipitates (lane 3, top and bottom panels). These biochemical data indicate that Tax associates with IKKgamma in the context of a high molecular mass Ikappa B kinase in HTLV-1-infected T cells.

To address the stability of these higher order Tax·IKKgamma complexes, IKKgamma was immunopurified from SLB-1 fractions containing peak kinase activity (>700 kDa) and washed at high stringency with escalating concentrations of NaCl and urea. We then monitored the dissociation of IKKalpha ·IKKbeta and Tax from these IKKgamma immunocomplexes using in vitro kinase and immunoblotting assays, respectively. As shown in Fig. 2D (bottom panel), high concentrations of either dissociation agent failed to release Tax from IKKgamma . Interactions between IKKgamma and the IKKalpha ·IKKbeta catalytic subunits were also highly resistant to release, as inferred from our ability to detect significant levels of IKKgamma -associated Ikappa B kinase activity under identical washing conditions (Fig. 2D, top panel). These results confirm that Tax, IKKgamma , and the catalytic subunits of IKK interact with high affinity, further underscoring the specificity and pathologic relevance of this viral/host interaction in HTLV-1-infected T cells.

IKKgamma Mediates the Functional Interaction between Tax and IKKbeta -- Tax activates TNF-responsive IKKs primarily via its stimulatory effects on IKKbeta (11), which interacts directly with IKKgamma (13-15). To determine whether IKKgamma directs the assembly of Tax·IKK complexes, mammalian 293T cells were transfected with expression vectors for FLAG-tagged IKKbeta , IKKgamma , and Tax. Cytoplasmic extracts were then prepared and subjected to immunoprecipitation with either monoclonal anti-FLAG antibodies (Fig. 3A, top and middle panels) or Tax-specific antibodies (Fig. 3A, bottom panel). When IKKbeta immunocomplexes were probed on immunoblots for the presence of Tax, we found that IKKbeta interacted weakly with Tax-WT, Tax-M22, and Tax-M47 in IKKgamma -deficient cells (Fig. 3A, lanes 2, 5, and 8, top panel). In contrast, significant amounts of both Tax-WT and Tax-M47 were associated with IKKbeta in cells coexpressing IKKgamma (lanes 3 and 9). Under identical transfection conditions, Tax-M22 failed to interact appreciably with IKKbeta in the presence of ectopic IKKgamma (lane 6). This divergent result with Tax-M22 could not be attributed to inefficient ectopic expression, because comparable amounts of IKKgamma and Tax protein were detected in each triple transfection (Fig. 3A, lanes 3, 6, and 9, middle and bottom panels). Coupled with our prior observation that Tax-M22 is defective for binding to endogenous IKKs (12), these data strongly suggest that IKKgamma confers IKKbeta targeting specificity to Tax.


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Fig. 3.   IKKgamma directs the formation of active Tax·IKKbeta complexes. A, 293T cells (1 × 106) were cotransfected with expression plasmids for FLAG-tagged IKKbeta (1 µg), Myc-tagged IKKgamma (0.5 µg), and Tax (2 µg) as indicated. Ectopic IKKbeta and Tax were isolated from cytoplasmic extracts by immunoprecipitation (IP) with anti-FLAG M2 (top and middle panels) and anti-Tax (lower panel) antibodies, respectively. Resultant immunocomplexes were analyzed for the presence of either Tax or IKKgamma on immunoblots as indicated in the right margin. The position of immunoglobulin heavy chains (H) is indicated. B, S107 cells (1 × 107) were cotransfected with expression vectors for FLAG-tagged IKKbeta , IKKgamma , and Tax (5 µg each) as indicated. Ectopic IKKbeta was immunoprecipitated from cytoplasmic extracts as described above and assayed for Ikappa B kinase activity (see Fig. 2A, legend). WT, wild type.

To explore the functional consequences of these higher order interactions, expression vectors for IKKbeta , IKKgamma , and Tax were introduced into S107 plasmacytoma cell line. Importantly, S107 cells harbor a genetic defect that impairs NF-kappa B expression (23), thus providing a cellular background with minimal Ikappa B kinase activity. Following transfection, ectopic IKKbeta was immunopurified from S107 cytoplasmic extracts and monitored for catalytic activity using an in vitro kinase assay. As shown in Fig. 3B, all three of the Tax constructs failed to stimulate IKKbeta kinase activity in the absence of ectopic IKKgamma (lanes 2-4), whereas overexpression of IKKgamma in cells harboring wild type Tax and Tax-M47 potently induced IKKbeta (lanes 6 and 8). In contrast, Tax-M22 was unable to activate IKKbeta in the presence of IKKgamma (lane 7), consistent with its defect in endogenous IKK binding (12). These functional data correlate precisely with our biochemical results demonstrating that IKKgamma directs the formation of Tax-M47·IKKbeta but not Tax-M22·IKKbeta complexes in mammalian 293T cell transfectants (Fig. 2B).

The N Terminus of IKKgamma Is Required for Tax Targeting to IKKbeta -- Primary sequence analyses indicate that IKKgamma contains a C-terminal leucine zipper domain, a central coiled-coil domain, and an N-terminal domain with no apparent secondary structural features (13-16). To define the sequences in IKKgamma that mediate its Tax adaptor function, Tax and FLAG-tagged IKKbeta were transiently expressed in 293T cells along with a panel of Myc epitope-tagged deletion mutants of IKKgamma (Fig. 4A). Cytoplasmic extracts were prepared from transfected cells and subjected to immunoprecipitation with antibodies specific for each ectopic protein. The resultant immunocomplexes were then probed for the presence of Tax and IKKgamma protein on immunoblots. As shown in Fig. 4B, removal of C-terminal sequences either abutting or encompassing the leucine zipper domain of IKKgamma (mutants D1 and D2) had no detectable effect on the formation of IKKbeta ·Tax and IKKbeta ·IKKgamma complexes (lanes 4 and 5, top and middle panels). However, deletion of the N-terminal region of IKKgamma (amino acids 1-100, mutant D3) completely disrupted both of these interactions (lane 6, top and middle panels). We consider these results to be significant, because the three IKKgamma deletion mutants were comparably coexpressed with Tax in the cytoplasmic compartment (Fig. 4B, lower panels). Furthermore, parallel experiments conducted with Tax-M22 confirmed the specificity of these higher order interactions (Fig. 4B, lanes 7-12).


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Fig. 4.   The N terminus of IKKgamma is required for Tax·IKK targeting. A, schematic representation of full-length and truncated forms of IKKgamma . Relative positions of the N-terminal region (N, closed boxes), coiled-coil domain (shaded boxes), leucine zipper (LZ, striped boxes), and C-terminal region (C, open boxes) are shown. Designations and deletion end points for each Myc-tagged mutant are indicated. aa, amino acids. B, 293T cells (3 × 106) were cotransfected with expression plasmids for FLAG-tagged IKKbeta (3 µg), Tax (6 µg), and the indicated Myc-tagged forms of IKKgamma (1.5 µg). Proteins indicated in the left margin were isolated from cytoplasmic extracts by immunoprecipitation (IP) with antibodies specific for FLAG (top two panels), Myc (third panel), or Tax (lower panel) epitopes. Resultant immunocomplexes were probed on immunoblots with either Tax- or IKKgamma -specific antibodies as indicated in the right margin. C, 293T cells (1 × 106) were cotransfected with expression plasmids for Tax (4 µg) and Myc-tagged forms of either wild type (WT) or truncated IKKgamma as indicated (4 µg each, see panel A). Ectopic IKKgamma was immunoprecipitated from cytosolic extracts using agarose-coupled anti-Myc antibodies. Resultant immunocomplexes were monitored for either IKK activity (top panel) or IKKgamma protein levels (bottom panel) using the in vitro kinase assay and immunoblotting procedures described in Fig. 2.

To extend these findings with ectopically expressed IKKbeta , we monitored the incorporation of the same IKKgamma mutants into endogenous IKK complexes. For these studies, 293T cells were transfected with IKKgamma and Tax expression vectors, followed by immunoprecipitation of ectopic IKKgamma from the corresponding cytoplasmic extracts. The resultant IKKgamma immunocomplexes were analyzed for the presence of Tax-activated IKKs using an in vitro kinase assay. As shown in Fig. 4C (top panel), overexpression of wild type IKKgamma alone yielded minimal endogenous IKK activity (lane 2). However, coexpression of Tax with IKKgamma led to potent activation (lane 3). Consistent with their ability to target Tax to ectopic IKKbeta , IKKgamma mutants D1 and D2 were fully competent to mediate Tax activation of endogenous IKK catalytic activity (lanes 4 and 5). In contrast, the N-terminal deletion mutant of IKKgamma failed to reconstitute a functional Tax·IKK signaling axis (lane 6). We conclude that the N terminus of IKKgamma is required for stable integration of this subunit into endogenous IKK, which in turn renders the holoenzyme susceptible to persistent activation by Tax.

In summary, we have found that the IKKgamma subunit of TNF-responsive IKKs is an essential core component of Tax-associated IKKs in HTLV-1-infected T cells. Higher order complexes containing Tax, IKKgamma , and the IKKalpha ·IKKbeta catalytic subunits are highly resistant to dissociation in vitro, underscoring the specificity of this pathologic viral/host interaction. In vivo reconstitution experiments demonstrate that IKKgamma directs the assembly of Tax·IKKbeta complexes, resulting in the persistent expression of Ikappa B kinase activity. Thus, IKKgamma functions in Tax-mediated IKK activation at the level of Tax·IKK docking. By analogy, this targeting mechanism may reflect an important role for IKKgamma in coupling TNF-responsive IKKs to upstream physiologic activators, such as NIK and MEKK1 (31).

    ACKNOWLEDGEMENTS

We thank Shoji Yamaoka, Alain Israel, Frank Mercurio, David Rothwarf, Michael Karin, and Brian Seed for reagents.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant RO1 AI33839 (to D. W. B), by NCI, National Institutes of Health Training Grant T32 CA09385 (to Z.-L. C.), and by the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Howard Hughes Medical Inst., Vanderbilt University School of Medicine, 802 Rudolph Light Hall, Nashville, TN 37232-0295. Tel.: 615-343-1548; Fax: 615-343-5743; E-mail: dean.ballard{at}mcmail.vanderbilt.edu.

    ABBREVIATIONS

The abbreviations used are: HTLV-1, human T cell leukemia virus type 1; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; IKK, Ikappa B kinase; TNF, tumor necrosis factor-alpha .

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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