©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Molecular Mechanism for Human T-cell Leukemia Virus Latency and Tax Transactivation (*)

Anne Brauweiler , Pamela Garl , Audrey A. Franklin , Holli A. Giebler , Jennifer K. Nyborg (§)

From the (1) Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human T-cell leukemia virus type I (HTLV-I) is the causative agent of an aggressive T-cell malignancy in humans. While the virus appears to maintain a state of latency in most infected cells, high level virion production is an essential step in the HTLV-I life cycle. The virally-encoded Tax protein, a potent activator of gene expression, is believed to control the switch from latency to replication. Tax stimulation of HTLV-I transcription is mediated through cellular activating transcription factor/cAMP response element binding proteins, which bind the three 21-base pair (bp) repeat viral enhancer elements. In this report, we show that viral latency may result from a highly unstable interaction between CREB and the HTLV-I 21-bp repeats, resulting in rapid dissociation of CREB from the viral promoter. In the presence Tax, the dissociation rate of CREB from a 21-bp repeat element is decreased. This stabilization is highly specific, requiring the amino-terminal region of CREB and appropriate 21-bp repeat sequences. We suggest that Tax stabilization of CREB binding to the viral promoter leads to an increase in gene expression, possibly providing the switch from latency to high level replication of the virus.


INTRODUCTION

Human T-cell leukemia virus type 1 (HTLV-I)() is the retrovirus found to be the causative agent of adult T-cell leukemia, an aggressive and fatal malignancy of T-helper cells (for review, see Ref. 1). It is estimated that less than 4% of HTLV-I-infected individuals develop adult T-cell leukemia, with disease onset occurring several decades after infection (2-4). The virus is believed to establish and maintain a state of latency in the infected T-cell, with very low levels of viral gene expression (5, 6) . A critical step in the life cycle of the virus is the transition from the latent infection to high levels of viral gene expression, resulting in virion production. The key viral regulatory protein, which may control this switch, is the HTLV-I oncoprotein Tax. Tax stimulates transcription of HTLV-I through interaction with host cell transcription factors rather than through direct binding to the viral promoter DNA (7, 8, 9, 10) . These cellular transcription factors mediate Tax transcriptional activation through three 21-bp repeat enhancer elements located in the transcriptional control region of the virus (11, 12, 13, 14) . Each 21-bp repeat contains a core nucleotide sequence with similarity to the palindromic cAMP response element (CRE). The CRE's are recognition sites for members of the ATF/CREB family of basic leucine zipper (bZIP) transcription factors (15) . The ATF/CREB proteins have been widely studied as mediators of both viral gene expression and Tax transactivation (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) . We have previously shown that two members of this family, CREB and ATF-2, are the principal T-cell proteins that directly bind the HTLV-I 21-bp repeats, and activate transcription in vitro(24) .

A significant number of studies support a role for the ATF/CREB proteins in the mediation of transcriptional stimulation by Tax; however, the precise molecular mechanism of Tax transactivation is incompletely understood. Several recent studies provided evidence suggesting that Tax stimulates viral transcription through enhancement in the DNA binding of cellular activator proteins, including CREB and ATF-2, to the 21-bp repeats (22, 23, 24, 26, 29, 30) . This Tax-dependent enhancement in DNA binding activity appears to occur, in part, through an increase in bZIP protein dimerization (26, 30).() Other studies provide evidence for a distinct mechanism of Tax transactivation in which Tax stimulates transcription through direct tethering to the HTLV-I 21-bp repeats. In this model, Tax is anchored to the promoter via protein-protein interactions with the bound ATF/CREB proteins. This hypothesis is supported primarily by data demonstrating transcriptional activation properties intrinsic to the Tax protein (32, 33, 34, 35) .

To further characterize the mechanism of Tax transactivation, we used equilibrium binding and dissociation kinetics to study the interaction of CREB and ATF-2 with the 21-bp repeat sequences both in the presence and absence of Tax. We demonstrate in this report that the three 21-bp repeats represent lower affinity CRE sequences, consistent with a possible role for these elements in maintaining viral latency. We further demonstrate that Tax enhances the equilibrium binding affinity of both CREB and ATF-2 for the 21-bp repeats and a consensus CRE; however, only the 21-bp repeats conferred Tax transactivation to a heterologous promoter in vivo. Finally, we provide two independent lines of evidence showing that Tax transactivation may result directly from Tax stabilization of CREB, and not ATF-2, binding to the 21-bp repeats. This Tax-dependent stabilization was dependent upon specific nucleotide sequences located within the 21-bp repeat element and upon amino acids in the amino-terminal region of CREB.

Together, these data support a model in which viral latency results from the presence of off-consensus CRE sequences in the viral 21-bp repeats. These lower affinity sequences enable rapid dissociation of CREB from the HTLV-I promoter and therefore fail to support transcriptional activation. In the presence of Tax, however, the transition from viral latency to high level expression is achieved via a highly specific interaction between Tax, CREB, and the 21-bp repeat element. This interaction results in significant stabilization of CREB binding to the 21-bp repeat sequences followed by transcriptional activation of the HTLV-I genome.


EXPERIMENTAL PROCEDURES

Recombinant Plasmids, Cell Culture, and Transfections

The reporter plasmid pminCAT (formerly called pGLCAT, see Ref. 36) was used as the parent vector for cloning double-stranded oligonucleotides representing the CRE and CRE-like elements given in the text (see I). Three copies of each element were cloned into pminCAT immediately upstream of the herpesvirus thymidine kinase promoter TATA box, at position -35 relative to the start site of transcription. The final constructs were confirmed by DNA sequencing. The reporter plasmids were tested in the presence and absence of the Tax expression plasmid, HTLV-I Tax (14) . Transient co-transfection assays were performed in CV-1 cells, grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mML-glutamine, and antibiotics, with 12 µg of DNA total. CV-1 cells were used in the transfection assay, as they have been widely used to demonstrate Tax transactivation. The percent of acetylated C-labeled chloramphenicol was determined by PhosphorImager analysis.

Expression and Purification of CREB, ATF-2, and Tax

CREB (amino acids 1-327), truncated CREB (amino acids 254-327), and ATF-2 (amino acids 1-505) were expressed in Escherichia coli and purified by the methods described previously (37, 24) followed by heparin agarose chromatography. All proteins were purified to greater than 95% homogeneity. We have previously demonstrated sequence-specific DNA binding of each protein (24) . Tax was expressed in E. coli from the pTaxH expression vector (20) and purified to apparent homogeneity by Ni chelate chromatography followed by gel filtration (see Ref. 23). The Tax concentration ranged between 40 and 80 ng/µl. Mock Tax was prepared from E. coli cells transformed with a control plasmid not encoding the Tax gene. This mock preparation of Tax was expressed, purified, and analyzed exactly as described for wild-type Tax (see Ref. 24).

Electrophoretic Mobility Shift Assays (EMSA)

Protein-DNA interactions were studied by incubation of the purified proteins with the appropriate P-end labeled DNA probes. The binding reactions contained the DNA probe, 12.5 mM HEPES, pH 7.9, 6.25 mM MgCl, 5 µM ZnS0, 75 mM KCl, 2 mM -mercaptoethanol, 10% (v/v) glycerol, 0.05% (v/v) Nonidet P-40, and the indicated concentrations of purified CREB, truncated CREB, or ATF-2, and/or Tax in a 20-µl sample. Reactions were incubated at room temperature and analyzed on 5.2% nondenaturing polyacrylamide gels (49:1, acrylamide/N,N-methylenebisacrylamide). The electrophoresis buffer contained 0.025 M Tris, 0.189 M glycine, pH 8.5, and 0.1% Nonidet P-40. Gels were dried, autoradiographed, and quantitated by PhosphorImager analysis.

Equilibrium and Kinetic Binding Studies

For the equilibrium binding studies, we ensured that the concentration of free protein approximated the total protein concentration by keeping the amount of labeled DNA probe constant at a level below 0.05 nM. Time course studies, performed for both CREB and ATF-2, were used to establish when DNA binding reactions reached equilibrium (data not shown). Both bound and free probe were quantitated to determine the percent of DNA complexed with protein. The concentration of active protein was determined by the method of DNA titration (38) and adapted for the electrophoretic mobility shift assay. The apparent K values were determined as described previously (38) . Computer analysis of the data was performed with the KaleidaGraph software program. Comparison of equilibrium binding curves produced by parallel EMSA and DNase I footprinting assays showed essentially identical results (data not shown). Inclusion of 100-fold excess of nonspecific competitor DNA or 1 µg of BSA in the binding reactions had no effect on the binding curve in the presence or absence of Tax (data not shown). For the dissociation kinetic studies, the third 21-bp repeat or consensus CRE probes were incubated with a concentration of CREB to produce approximately 50% of the probe bound in complex. The binding reactions were allowed to reach equilibrium and then challenged with a 1000-fold molar excess of unlabeled homologous binding site. The samples were loaded onto a running gel at appropriate times following challenge and resolved by EMSA, and the percent bound was determined as described above.

DNA Probes

Complimentary double-stranded oligonucleotides were cloned into the BamHI site in the polylinker of pUC19, and the 80-bp EcoRI-HindIII fragments were purified and P-5`-end labeled for use as probes in the EMSA reactions. These larger cloned fragments were more stable than synthetic oligonucleotide probes, enabling accurate quantitation. DNA fragment concentrations were determined by UV absorbance at 260 nm. The nucleotide sequence of the binding sites used in this study are reported (see I).


RESULTS

The Determination of CREB and ATF-2 DNA Binding Affinities in the Absence and Presence of Tax

We have previously identified CREB and ATF-2 as the principal T-cell proteins that bind the three 21-bp repeats in the HTLV-I promoter and stimulate HTLV-I transcription in a cell-free extract (24) . The CRE-like sequences present within each 21-bp repeat share similarity, but not identity, with the consensus palindromic CRE sequence, TGACGTCA. Because the initiation of transcription depends upon the stable binding of transcription factors to specific DNA elements, we were interested in determining whether the binding affinities of CREB and ATF-2 for the HTLV-I 21-bp repeat elements were significantly lower than for a consensus CRE.

For the initial experiment, we performed an equilibrium binding assay to examine CREB binding affinity to a consensus CRE sequence from the human chorionic gonadotropin gene (hCG) promoter (Fig. 1A). In this experiment, the amount of labeled CRE DNA was kept constant, and purified recombinant CREB protein was varied over a 100-fold concentration range. The binding reactions were allowed to reach equilibrium, and the protein-DNA complexes were analyzed by EMSA. Several protein-DNA complexes were observed. The major complex, observed at lower CREB concentrations, represented a single CREB homodimer bound to the CRE probe; the slower migrating complexes observed at high CREB concentrations likely represented multimeric forms of CREB bound to the 80-bp probe. All complexes were equally competed by a 100-fold excess of unlabeled consensus CRE oligonucleotide, whereas an unrelated competitor DNA had no effect, indicating that CREB bound specifically to the CRE (data not shown). To determine the apparent binding affinity, the fraction of CRE probe bound versus the total CREB concentration was plotted (Fig. 1B). The concentration of protein required for half-maximal DNA saturation (K) was used to determine the apparent affinity of CREB for the CRE. The affinities are reported as K as the shape of the curve suggests multistep binding. Analysis of the midpoint of binding indicated an apparent K of CREB binding to the hCG consensus CRE of approximately 0.6 nM.


Figure 1: Equilibrium binding of CREB to a consensus CRE sequence. A, protein titration experiment analyzed by EMSA. Equilibrium binding reactions contained a constant amount of CRE probe and the indicated concentration of CREB monomer. The positions of protein-DNA complex and free probe are indicated. B, a graph showing the fraction of CRE probe bound, from EMSA shown in A, plotted as a function of CREB concentration. The dashedline is the best fit to the data.



Equilibrium binding titrations were performed as above to determine the relative affinity of both ATF-2 and CREB for the three 21-bp repeats. The result of these studies are summarized in . For comparison, the affinity for the consensus CRE is also reported. As indicated in , the CRE-like sequences present in the 21-bp repeats of HTLV-I each contain one or more base changes from the consensus CRE sequence. These base changes likely contribute to the approximately 10-fold lower affinity observed for each of the three 21-bp repeats, relative to a consensus CRE site. Interestingly, both CREB and ATF-2 demonstrated parallel reduced affinity for the three 21-bp repeats.

The DNA binding activities of CREB and ATF-2 (and other bZIP proteins) are dramatically enhanced in the presence of the viral Tax protein (22-24, 26). To determine whether enhancement of DNA binding activity reflects a change in binding affinity, quantitative equilibrium binding studies were performed with ATF-2 and the third 21-bp repeat probe in the presence of highly purified recombinant Tax protein (Fig. 2A). Analysis of a plot of the binding data obtained in the presence and absence of Tax showed a large difference in both the concentration range and affinity of ATF-2 binding (Fig. 2B). In the presence of Tax, the apparent affinity of ATF-2 for the 21-bp repeat was increased, as the concentration required to bind half of the DNA changed from 12 nM in the absence of Tax to approximately 2.5 nM in the presence of Tax (Fig. 2B). In addition, ATF-2 bound over an approximately 10-fold greater range of protein concentration in the presence of Tax (Fig. 2, A and B). reports the affinities of CREB and ATF-2 for the third 21-bp repeat and the consensus CRE in the presence of Tax; in each case, Tax increased the equilibrium binding affinities between 5- and 10-fold.


Figure 2: Equilibrium binding of ATF-2 to the third 21-bp repeat in the presence and absence of Tax. A, protein titration analyzed by EMSA. Equilibrium binding reactions contained a constant amount of the third 21-bp repeat probe and the indicated amount of ATF-2 monomer. 100 nM Tax was added to the indicated reactions. The positions of protein-DNA complex and free probe are indicated. B, a graph showing the fraction of the third 21-bp repeat probe bound, from EMSA shown in A, plotted as a function of ATF-2 concentration. ATF-2 binding in the absence of Tax (diamonds) and in the presence of Tax (triangles) is shown.



To characterize the concentration dependence of Tax on bZIP protein binding, we measured the affinity of CREB to a 21-bp repeat in the presence of incremental changes in Tax protein concentration. Increasing concentrations of Tax produced a corresponding incremental decrease in apparent K of CREB for the third 21-bp repeat element (Fig. 3). Together, these data indicate that the mechanism of Tax enhancement of CREB and ATF-2 DNA binding involves a Tax concentration-dependent increase in the affinity of the bZIP proteins for their CRE and CRE-like recognition elements.


Figure 3: The change in affinity of CREB for a 21-bp repeat is directly proportional to the concentration of Tax. Equilibrium binding reactions, containing a constant amount of the third 21-bp repeat probe, varying amounts of Tax, and the indicated amount of CREB monomer, were assayed by EMSA, and the results were plotted as described in Fig. 1B. CREB binding in the absence of Tax (diamonds) and in the presence of 200 ng (circles) and 400 ng (triangles) of Tax is shown.



Correlation between DNA Binding Affinities and Transcriptional Activation in the Absence and Presence of Tax

Since enhancement in DNA binding activity may represent a mechanism of Tax transactivation, we were interested in determining whether a correlation existed between the in vitro binding affinities, obtained in both the presence and absence of Tax, and basal and Tax-induced transcriptional activation in vivo. To test this, we constructed heterologous promoters containing three copies of either the third 21-bp repeat or the consensus CRE, cloned immediately upstream of the herpesvirus thymidine kinase minimal promoter linked to the CAT gene. We focused our study on the third, most promoter-proximal, 21-bp repeat, as comparable binding affinities were obtained for all three 21-bp repeat elements. The chimeric constructs were transfected into CV-1 cells in the presence and absence of a Tax expression vector. The HTLV-I promoter, LTR-CAT, and the parent construct, pminCAT, were tested in parallel as positive and negative controls, respectively. The results of the CAT assay are presented in Fig. 4 . In the absence of Tax, the consensus CRE-containing construct produced an approximately 10-fold higher CAT activity relative to the third 21-bp repeat construct (compare lanes9 and 10 with lanes13 and 14). The third 21-bp repeat construct conferred no increase in CAT activity over the parent vector (compare lanes5 and 6 with lanes 13and 14). This difference between the 21-bp repeat and the consensus CRE constructs correlated nicely with the approximately 10-fold difference in CREB and ATF-2 binding affinities observed between these two sites in the in vitro binding assays. This strong correlation between the in vitro binding data and in vivo functional data suggest that CREB and/or ATF-2 play a role in basal expression of the virus in the cell. In the presence of Tax, however, we observed over a 40-fold increase in CAT activity with the third 21-bp repeat construct (Fig. 4, lanes13-16), with no effect of Tax on the consensus CRE-CAT construct (Fig. 4, lanes9-12). Tax transactivation mediated through the 21-bp repeat was dependent upon wild-type Tax, as transfection of a mutant Tax construct had no effect (data not shown). These in vivo results, obtained in the presence of Tax, contrast with our equilibrium binding data, as we observed similar Tax-dependent increases in DNA binding activity in vitro with both the third 21-bp repeats and the consensus CRE.


Figure 4: Comparison of Tax transactivation mediated through the third 21-bp repeat and consensus CRE. Transient co-transfection assays were performed in CV-1 cells with 10 µg of the indicated CAT reporter plasmids in the absence or presence of 2 µg of the HTLV-I-Tax expression plasmid (14) as indicated (lanes5-16). As a positive control, pU3RCAT (2 µg), the HTLV-I LTR CAT reporter plasmid, was assayed in the presence and absence of Tax (lanes1-4). The percent conversion to acetylated [C]chloramphenicol together with the -fold activation in the presence of Tax are shown in the figure. The -fold activation is based on the average of the duplicates. Although the figure represents the results of a single experiment, similar Tax activation was observed in four independent experiments. A schematic representation of the CAT reporter plasmid (pminCAT), carrying three copies of the third 21-bp repeat (pminCAT-21-bp repeat) or consensus CRE (pminCAT-CRE) cloned upstream of the thymidine kinase proximal promoter is shown.



Dissociation Kinetics Reveal a Highly Specific Tax Stabilization of CREB Binding to a 21-bp Repeat

To investigate the molecular basis for the dramatic difference between Tax transactivation mediated by the third 21-bp repeat and the consensus CRE, we analyzed the dissociation kinetics of CREB binding to these sites in vitro. Probes representing either the third 21-bp repeat or the consensus CRE were incubated with CREB in the presence or absence of Tax. Binding reactions were then challenged with a large excess of unlabeled DNA binding site, and the kinetics of dissociation were determined by quantitative analysis of the EMSA. A plot of CREB dissociation from each DNA sequence is shown in Fig. 5. In the absence of Tax, the dissociation of CREB from the third 21-bp repeat was very rapid (t 30 s), whereas CREB binding to the consensus CRE was significantly more stable (t = 17 min). The presence of Tax in the reaction significantly stabilized the binding of CREB to the third 21-bp repeat, whereas Tax had little or no effect on CREB binding to the consensus CRE. Surprisingly, we did not observe Tax stabilization of the binding of ATF-2, or a truncated form of CREB, containing principally the DNA binding and dimerization domain of the protein on either the consensus CRE or the 21-bp repeat sequence (data not shown). These data indicate that the effect of Tax on dissociation kinetics is specific for full-length CREB binding to a 21-bp repeat. Interestingly, the Tax-dependent stabilization of CREB on the 21-bp repeat, and not the consensus CRE, correlates precisely with the in vivo Tax transactivation results presented above. Tax has also been shown to stimulate the rate of bZIP protein association for DNA (26) . Together, these data suggest that the effect of Tax on the dissociation rate, and not the association rate, contributes to Tax transactivation of HTLV-I in vivo.


Figure 5: Kinetics of CREB dissociation in the presence and absence of Tax. Tax prevents dissociation of CREB from a 21-bp repeat but not to the consensus CRE. Purified, recombinant CREB was incubated with either the consensus CRE or the third 21-bp repeat probe in the absence or presence of purified Tax. Equilibrium binding reactions were then challenged with a 1000-fold molar excess of the unlabeled binding site, and the kinetics of dissociation were analyzed by EMSA (see ``Experimental Procedures''). CREB dissociation was quantitated, and the concentration of CREB, remaining bound relative to the concentration bound at time zero (no added competitor) (B/B), was plotted as a function of time following challenge. CREB dissociation from either the third 21-bp repeat probe in the absence (closedtriangles) and presence (closedcircles) of Tax or the consensus CRE probe in the absence (opentriangles) and presence (opencircles) of Tax is shown.



The strong correlation showing a dependence upon the 21-bp repeat element for both Tax stabilization of CREB binding and Tax transactivation in vivo prompted us to test whether the critical nucleotide sequences lie within the 21-bp CRE-like octanucleotide core or within the DNA sequence adjacent to the core. To test this hypothesis, we prepared the two hybrid binding sites shown in I. The first contains the off-consensus CRE-like core sequence (TGACGACA) from the third 21-bp repeat, with adjacent flanking sequences derived from the hCG consensus CRE site (site 3 core/CRE). The second contains the consensus CRE core sequence (TGACGTCA), with adjacent flanking sequences derived from the third 21-bp repeat (CRE core/site 3). We measured the dissociation rate of CREB binding to these hybrid sequences in the presence and absence of Tax (Fig. 6, A and B). Examination of the kinetic data indicates that the core CRE octanucleotide, whether consensus or off-consensus, had no effect on Tax-dependent stabilization. The sequences that directly flank the core, however, had a dramatic effect on CREB binding stability in the presence of Tax.


Figure 6: Tax stabilization of CREB binding is dependent upon 21-bp sequences adjacent to the CRE octanucleotide core. Plot of the dissociation kinetic data was performed and analyzed exactly as described for Fig. 5, except CREB was assayed in the absence (closedcircles) and presence (opentriangles) of Tax with the following probes: A, the off-consensus CRE-like core sequence from the third 21-bp repeat, with adjacent flanking sequences derived from the hCG consensus CRE site (site 3 core/CRE) (see Table III for sequence); B, the consensus CRE core sequence, with adjacent flanking sequences derived from the third 21-bp repeat (CRE core/site 3) (see Table III for sequence).



Correlation between Tax-dependent Changes in Dissociation Kinetics and Tax Transactivation in Vivo

Our observation that the DNA sequences immediately adjacent to the core octanucleotide promoted specific stabilization of CREB binding prompted us to test whether these same DNA sequences may confer increased Tax transactivation in vivo. To test this hypothesis, we constructed heterologous promoters containing three copies of the two hybrid binding sites (I). The binding sites were cloned into pminCAT as described above. The chimeric constructs were transfected into CV-1 cells in the presence and absence of a Tax expression vector. The results of the CAT assay are presented in Fig. 7 . In surprising agreement with the in vitro dissociation rate data, we found that the construct carrying the consensus CRE core and the 21-bp repeat flanking sequence (CRE core/site 3) conferred Tax transactivation, whereas the construct carrying the 21-bp repeat core and CRE flanking sequence was unresponsive (Fig. 7). These data confirm that the sequences that immediately flank the 21-bp repeat CRE-like core were critical in conferring Tax transactivation. This CAT assay data correlates precisely with the dissociation kinetic data presented above and provides strong support for a model of Tax transactivation occurring through the specific stabilization of CREB binding a 21-bp repeat.


Figure 7: Identification of 21-bp repeat nucleotide sequence elements required for Tax transactivation. Transient co-transfection assays were performed in CV-1 cells using 10 µg each of the pminCAT reporter plasmids with three copies of the indicated cloned hybrid binding sites (see Table III for sequence). Transfections were performed in the absence or presence of 2 µg of the HTLV-I-Tax expression plasmid, as indicated. The percent conversion to acetylated [C]chloramphenicol together with the -fold activation in the presence of Tax are shown in the figure. The -fold activation is based on the average of the duplicates. This experiment is a continuation of the data shown in Fig. 4 (see Fig. 4 for controls).



Alterations in the Mobility of EMSA Complexes Further Support a Role for CREB and a 21-bp Repeat in Mediating Tax Transactivation

Our demonstration that Tax stabilized full-length CREB binding in a DNA sequence-dependent manner prompted us to directly compare CREBDNA complexes formed with each probe by EMSA. Labeled probes representing the third 21-bp repeat, a consensus CRE, and the two hybrid sites (see I) were compared in binding assays with CREB in the presence or absence of Tax (Fig. 8A). In all cases, Tax enhanced the DNA binding activity of CREB to the labeled probes as expected. However, examination of the CREBDNA interaction in the presence of Tax revealed differences in complex mobility in a probe-dependent manner. Tax significantly reduced the mobility of the CREBDNA complex obtained with the third 21-bp repeat probe, but had only a modest effect on the mobility of the complex formed with the consensus CRE (Fig. 8A, lanes1-8). In addition, comparison of the two hybrid sites shows that flanking sequences derived from the third 21-bp repeat were responsible for this reduced mobility complex (Fig. 8A, lanes9-16). A mock preparation of Tax, which was expressed, purified, and analyzed in parallel with wild-type Tax, did not produce enhancement in CREB DNA binding activity or alterations in the mobility of the CREBDNA complex (Fig. 8B). We also did not observe Tax-dependent changes in the EMSA migration of the ATF-2-DNA complex (data not shown).


Figure 8: A, Tax alters the mobility of CREBDNA complexes in a sequence-dependent manner. 10 ng of purified, recombinant CREB was incubated with probes representing sequences from the consensus CRE (lanes1-4) and the third 21-bp repeat (lanes5-8). The hybrid binding sites (see Table III) representing the consensus CRE core/third 21-bp repeat flank (CRE core/site 3) (lanes9-12), and the third 21-bp repeat CRE-like core/consensus CRE flank (site 3 core/CRE) (lanes13-16) were tested in parallel. Binding reactions were performed in the absence or presence of the indicated volume of purified Tax and analyzed by EMSA. The position of the free probe is indicated. B, mock Tax, expressed and purified exactly as wild-type Tax from E. coli, does not alter the mobility of the CREBDNA complex. Reaction mixtures contained the third 21-bp repeat probe, 10 ng of purified recombinant CREB, and the indicated amount of mock Tax or authentic Tax, as indicated. Binding reactions were analyzed by EMSA. The position of the free probe is indicated.



To delineate specific regions of CREB that might contribute to complex formation, we also tested the truncated form of CREB containing the DNA binding/dimerization domain (carboxyl-terminal 73 amino acids; positions 254-327). Fig. 9 shows that under conditions where full-length CREB binding to the third 21-bp repeat in the presence of Tax produced a complex with reduced mobility, truncated CREB was unaffected (compare lanes3-5 with lanes 6-8). The migration of both full-length and truncated CREB were unaffected by Tax on the consensus CRE probe (Fig. 9, lanes9-16). This observation was further supported by the absence of an effect of Tax on the dissociation rate of truncated CREB from the third 21-bp repeat probe (data not shown). Furthermore, the gel shift migration of complexes formed with the labeled 21-bp repeat probe and additional bZIP proteins (including ATF-1, truncated, and full-length forms of ATF-2, and Jun) were unaffected by Tax (data not shown). These data indicate that the association between Tax and the CREBDNA complex occurs only with appropriate DNA sequences and full-length CREB protein. Furthermore, the data implicate amino acids amino-terminal to the DNA binding-dimerization domain in the interaction.


Figure 9: Amino acids amino-terminal to the bZIP region of CREB are required to support interaction with Tax. 2 ng of either full-length CREB (amino acids 1-327) or truncated CREB (CREB BR) (amino acids 254-327) were incubated in the presence of the indicated amount of Tax protein. Identical reactions were performed with the third 21-bp repeat (lanes1-8) or consensus CRE probes (lanes9-16), as indicated. The protein-DNA complexes were analyzed by EMSA. The position of free probe is indicated.



To further examine the Tax-dependent alteration in complex mobility, we performed a Tax titration experiment. We reasoned that if the reduced mobility complex represented a stable stoichiometric association of Tax with CREB and DNA, as has been reported previously (27), a single ``supershifted'' gel shift complex of a discrete size should be observed. Furthermore, at lower Tax concentrations, two bands should appear; one representing the ternary complex and the second representing the CREBDNA complex without Tax. Fig. 10shows the result of this titration experiment. Surprisingly, the inclusion of increasing amounts of Tax in the gel shift reaction produced an incremental change in the mobility of the CREBDNA complex, with progression to a slower migrating, supershifted complex at higher Tax concentrations. These data support a weak or transient interaction between Tax and the CREBDNA complex, resulting in a dose-dependent reduction in complex mobility.


Figure 10: The degree of supershift is dependent upon the Tax concentration in the reaction. 2 ng of CREB were incubated in the presence of the indicated amount of Tax protein. Binding reactions were performed with the third 21-bp repeat probe, and the protein-DNA complexes were analyzed by EMSA. The positions of the protein-DNA complex (arrow) and free probe are indicated.




DISCUSSION

The three Tax-responsive 21-bp repeats serve as binding sites for members of the ATF/CREB family of transcription factors, including CREB and ATF-2. Since Tax does not bind the 21-bp repeats directly, it is generally believed that members of the ATF/CREB family mediate Tax transactivation. However, the precise mechanism by which Tax transactivates through these proteins is not understood. In this report, we examined the kinetic and equilibrium binding behavior of CREB and ATF-2 for various DNA sequences in the presence and absence of Tax. We then compared the in vitro binding data with in vivo Tax transactivation to correlate the functional significance of the data.

We demonstrated that, in the absence of Tax, the three 21-bp repeat elements located in the transcriptional control region of HTLV-I represent binding sites with modest affinity for CREB and ATF-2. There was a strong correlation between the in vitro binding affinities of the sites and their ability to confer transcriptional activation to a heterologous promoter in vivo. The lower affinity 21-bp repeats conferred essentially no increase in transcriptional activation, whereas the higher affinity consensus CRE increased the level of CAT activity approximately 10-fold. We conclude that the weaker 21-bp repeat elements may be essential to maintain viral latency, as high affinity CRE recognition elements would likely promote elevated levels of viral gene expression and prohibit the virus from escaping immune detection.

In the presence of Tax, both CREB and ATF-2 were transformed into higher affinity binding forms in vitro, with each protein demonstrating an approximately 10-fold higher affinity for the 21-bp repeats and a consensus CRE. It is interesting to note that the affinity of the third 21-bp repeat in the presence of Tax approached the affinity of the consensus CRE observed in the absence of Tax, suggesting that Tax converts the relatively weak 21-bp repeats into higher affinity binding sites in vivo. This Tax-dependent conversion of the 21-bp repeats into high affinity recognition elements may play a role in the transition from latency to transactivation.

Although the equilibrium binding studies indicated comparable Tax-dependent increases in CREB and ATF-2 binding affinity for both 21-bp repeats and the consensus CRE, we observed Tax transactivation only through the 21-bp repeat element. These data essentially exclude the possibility that Tax transactivation occurs through direct interaction of Tax with the bound ATF/CREB proteins at the promoter, as the high affinity consensus CRE should bind more CREB and thus tether more Tax, resulting in higher levels of transcriptional activation. These data also suggest that enhancement in DNA binding activity only partially explains Tax transactivation, as we observed a significant increase in the affinity of both CREB and ATF-2 for the consensus CRE sequence, yet the consensus CRE was ineffective in mediating Tax transactivation. One possible explanation for these data is that the Tax-dependent increase in ATF/CREB binding affinity for the consensus CRE sequence does not result in enhanced transcription, as the high affinity binding sites are fully occupied in the absence of Tax. The lower affinity 21-bp repeats, however, are unoccupied in vivo in the absence of Tax, and the Tax-dependent increase in ATF/CREB binding affinity results in saturation of these sites and subsequent transcriptional activation from 21-bp repeat containing promoters.

Although Tax enhanced the binding of both CREB and ATF-2 to the consensus CRE and 21-bp repeat elements, dissociation kinetic studies revealed a highly specific Tax stabilization of CREB binding to a 21-bp repeat element. We did not observe Tax stabilization of ATF-2 binding to any of the probes tested. Surprisingly, the 21-bp repeat sequences responsible for this Tax-dependent stabilization of CREB binding were not localized to the CRE-like core octanucleotide but rather to the nucleotide sequences located directly adjacent to the core. We observed remarkable concordance between our observations showing the importance of the sequences immediately adjacent to the octanucleotide core for CREB binding stabilization in vitro and the importance of these sequences in conferring Tax transactivation in vivo. Our results are consistent with several other studies demonstrating a functional significance for these 21-bp repeat sequences in Tax transactivation in vivo(28, 39, 40, 41) . The correlation between the in vivo significance of the flanking sequences for Tax transactivation together with our observation of the importance of these sequences in stabilizing CREB binding in the presence of Tax in vitro provide strong evidence for a molecular mechanism of Tax transactivation. It is interesting that the CRE core sequences, whether consensus or off consensus, were irrelevant in both Tax-dependent stabilization of CREB binding in vitro and Tax transactivation in vivo. From these data, we conclude that the core CRE-like sequences are necessary, but not sufficient, for Tax transactivation. In this manner, the virus has evolved the most efficient use of the LTR promoter by using low affinity CRE sequences to maintain viral latency while at the same time using the sequences flanking the CRE to mediate Tax transactivation.

The effect of Tax on the dissociation kinetics of CREB from the 21-bp repeat strongly suggests that Tax directly interacts with the CREBDNA complex to stabilize CREB binding. Further support for this hypothesis was provided by the Tax-dependent formation of CREB21-bp repeat complexes with reduced mobility in an EMSA. We observed these reduced mobility complexes with full-length CREB protein, but not with a truncated form of CREB containing only its DNA-binding/dimerization domain, again implicating amino acids in the amino-terminal region of the protein as critical in the interaction. Furthermore, the same 21-bp repeat nucleotide sequences were required for this interaction as were required for Tax transactivation in vivo and Tax stabilization of CREB binding in vitro, lending further support for the significance of these sequences in Tax-mediated transcriptional activation.

Although we provide strong evidence supporting a direct interaction between Tax and the CREB21-bp repeat complex, our evidence also suggests that the association of Tax is weak and/or transient. This conclusion is based primarily on our observation of Tax concentration-dependent changes in the position of the supershifted EMSA complex (Fig. 10). Our detection of Tax concentration-dependent incremental changes in the mobility of the CREBDNA complex is consistent with a rapid association-dissociation of Tax with the CREB21-bp repeat complex. This observation does not exclude the possibility that a stable ternary complex forms but dissociates under the conditions of our EMSA. We continue, however, to favor a model in which Tax forms a weak or transient interaction with the CREB21-bp repeat complex, as several additional lines of evidence support this interpretation. These include the absence of a Tax-specific protein-DNA interaction using either DNase I or methidium propyl EDTA:Fe(II) footprinting (data not shown and Ref. 28) or methylation interference (28) and, finally, failure to detect Tax-CREB interactions by affinity chromatography (data not shown and Ref. 31). While we conclude that the interaction is weak or transient, the relative strength of the Tax interaction remains very controversial, as evidence supporting a more stable ternary complex between Tax, a 21-bp repeat element, and 3 specific amino acids in the basic DNA binding region of CREB has previously been reported (27) .

It is of interest that Tax enhances the DNA binding activity of a wide range of transcriptional activator proteins to a wide range of DNA sequences, yet the change in dissociation rate kinetics was very specific for CREB and the 21-bp repeats. We (data not shown) and others (26) have demonstrated that the pleiotropic enhancement in DNA binding activity results from an increase in the association rate of bZIP protein binding to DNA. The increase in association rate reflects a decrease in binding energy resulting from an interaction between Tax and the basic-spacer region of the bZIP proteins. This increase in association rate likely accounts for a portion of the Tax-dependent increase in DNA binding affinity that we observe for CREB, with an additional contribution in binding stability derived from the decrease in dissociation rate. Taken together, these data indicate that Tax interacts with the basic-spacer segment of CREB to increase the association rate of CREB to the DNA sequence. This reaction is concurrent with, or immediately followed by, a second reaction in which Tax interacts with the amino-terminal region of CREB and nucleotides within the 21-bp repeat element to decrease the dissociation rate of CREB from the DNA. In the context of the HTLV-I promoter, both reactions appear necessary for efficient Tax transactivation. It is tempting to speculate, however, that the effect of Tax on the DNA association rate of a wide range of cellular transcription factors is responsible for the highly pleiotropic deregulation of cellular gene expression.

In summary, we provide both DNA binding and functional evidence to indicate that the interaction between the ATF/CREB proteins and the HTLV-I 21-bp repeats is highly unstable, resulting in rapid dissociation of the transcription factors from the promoter DNA. This unstable protein-DNA interaction does not support efficient transcriptional initiation and, therefore, likely contributes to viral latency. The HTLV-I Tax protein enters into a transient or metastable complex with CREB and the 21-bp repeat elements. This complex is highly specific, requiring amino acids in the amino-terminal region of CREB and appropriate nucleotide sequences flanking the CRE octanucleotide core sequence. The formation of this complex results in the stabilization of CREB binding to a 21-bp repeat, thus slowing dissociation of CREB from the viral promoter. We suggest that this interaction provides the switch from viral latency to Tax transactivation, with subsequent high level replication of the virus in vivo.

  
Table: Apparent affinities for CREB and ATF-2 binding to a consensus CRE and the three HTLV-I 21-bp repeats

The mismatches in the 21-bp repeats, relative to the consensus sequence, are underlined. The third 21-bp repeat is the most promoter proximal.


  
Table: Apparent affinities for CREB and ATF-2 DNA binding in the presence of Tax


  
Table: Nucleotide sequences of the consensus CRE, third 21-bp repeat, and hybrid binding sites

The CRE and CRE-like octanucleotide core sequences are shown in boldface.



FOOTNOTES

*
This work was supported by an American Cancer Society Junior Faculty Research Award JFRA 506 (to J. K. N.) and Public Health Service Grant CA-55035 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 303-491-0420; Fax: 303-491-0494; E-mail: jnyborg@vines.colostate.edu.

The abbreviations used are: HTLV-I, human T-cell leukemia virus type 1; bp, base pair; CRE, cAMP response element; CREB, CRE binding protein; ATF, activating transcription factor; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase.

A. M. Baranger, C. R. Palmer, M. K. Hamm, H. A. Giebler, A. Brauweiler, J. K. Nyborg, and A. Schepartz, submitted for publication.


ACKNOWLEDGEMENTS

We thank members of the department, especially Drs. Robert and A. Young Woody, for generous advice and support.


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