Characterization of a Nuclear Export Signal within the Human T Cell Leukemia Virus Type I Transactivator Protein Tax*

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I transactivator protein Tax plays an integral role in the etiology of adult T cell leukemia, as expression of Tax in T lymphocytes has been shown to result in immortalization. In addition, Tax is known to interface with numerous transcription factor families, including activating transcription factor/cAMP response element-binding protein and nuclear factor- (cid:1) B, requiring Tax to localize to both the nucleus and cytoplasm. In this report, the nucleocytoplasmic localization of Tax was examined in Jurkat, HeLa, and U-87 MG cells. The results reported herein indicate that Tax contains a leucine-rich nuclear export signal (NES) that, when fused to green fluorescent protein (GFP), can direct nuclear export via the CRM-1 pathway, as determined by leptomycin B inhibition of nuclear export. However, cytoplasmic localization of full-length Tax was not altered by treatment with leptomycin B, suggesting that native Tax utilizes another nuclear export pathway. Additional support for the presence of a functional NES has also been shown because the NES mutant Tax(L200A)-GFP localized to the nuclear membrane in the majority of U-87 MG cells. Evidence has also been provided suggesting that the Tax NES likely exists as a conditionally masked signal because the truncation mutant Tax (cid:2) 214-GFP localized constitutively

I-associated myelopathy/tropical spastic paraparesis (HAM/ TSP). The HTLV-I transactivator protein Tax plays an integral role in productive virus replication and disease progression. Tax is known to interact with a number of cellular signaling pathways and interfaces with numerous transcription factor families, including activating transcription factor/cAMP response element-binding protein (ATF/CREB) and NF-B (1). Specifically, Tax enhances CREB binding within the HTLV-I long terminal repeat, which in turn enhances transcription of viral mRNA (2)(3)(4)(5). With respect to the NF-B pathway, cytoplasmic Tax acts by directly binding the IKK-␥-subunit of the IB kinase inhibitor complex. This association induces the phosphorylation and degradation of IB-␣, the inhibitor of NF-B, thereby allowing the NF-B complex to migrate to the nucleus and induce gene expression (6 -8).
Although the exact events that lead to ATL have not been defined, it has been shown that Tax plays an integral role in this pathologic process (9). Expression of Tax in T lymphocytes has been shown to result in immortalization (10,11). Normal cells contain checkpoint pathways that prevent cellular division before any DNA damage or chromosomal abnormalities are repaired. Most cancerous cells, including cells and cell lines derived from individuals with ATL, have abnormalities in these checkpoint pathways. A number of molecular mechanisms have been proposed to account for the abnormalities observed in leukemic cells derived from ATL patients. Relevant to these observations, Tax has been shown to bind to the human homolog of MAD1, resulting in a loss of MAD1 function and the appearance of multinucleated giant cells (12). Additionally, HTLV-I-infected cells are resistant to microtubule inhibitors, a trait that is associated with the loss of MAD protein function (13). Interestingly, MAD1 and MAD2 are localized to the cytoplasm in HTLV-I-infected cells, as opposed to the nucleus, where they have been shown to accumulate in normal cells.
Although previous studies have indicated that Tax contains a nuclear localization sequence (NLS) at its amino terminus (amino acids 2-58) (14), very little is known concerning the cisand trans-acting elements and factors, respectively, involved in transport of Tax from the nucleus to the cytoplasm. Recently, an amino acid sequence involved in the export of proteins from the nucleus has been identified and characterized (15,16). This nuclear export signal (NES) usually contains a string of hydrophobic amino acids, including leucine and isoleucine residues (Fig. 1). In conjunction with the cis-acting NES, the cellular protein CRM-1 has also been shown to facilitate export of NES-containing proteins to the cytoplasm (17)(18)(19)(20). The intracellular distribution of a variety of cellular proteins involved in transcriptional regulation occurs via the CRM-1-mediated nuclear export pathway. Nuclear export of several proteins through this pathway is regulated by diverse molecular mechanisms such as protein-protein interactions and ubiquitination that facilitate binding of CRM-1 to the NES (21)(22)(23). Thus, if the nucleocytoplasmic distribution of Tax is controlled by CRM-1 or other nuclear export proteins, then the level of activity generated by these interactions and modifications can have substantial effects on the level of viral and cellular gene transcription.
Previous studies have indicated that Tax is able to shuttle between the nucleus and cytoplasm (24), suggesting that Tax interfaces with a nuclear export pathway. In support of these observations, we have demonstrated that Tax contains an NES between amino acids 188 and 202. Functional properties of the Tax NES have been examined in two cell lines: HeLa (cervical carcinoma), a cell line that has been traditionally used to study Tax localization; and U-87 MG, a cell line of astrocytic origin that has been shown to contain higher levels of Tax within the cytoplasm compared with the nuclear compartment (25,26). The results reported herein indicate that Tax contains a leucine-rich NES that, when fused to green fluorescent protein (GFP), can direct nuclear export via the CRM-1 export pathway, as determined by leptomycin B (LMB) inhibition of nuclear export. However, cytoplasmic localization of full-length Tax was not altered by treatment with LMB, suggesting that native Tax may utilize another nuclear export pathway. Interestingly, a Tax NES point mutation (L200A) within the context of full-length Tax resulted in a nuclear envelope localization of Tax in the majority of transfected U-87 MG cells. Evidence is also provided suggesting that the Tax NES within its native context may be a masked signal because the carboxyl-terminal truncation mutant Tax⌬214-GFP was shown to localize constitutively to the cytoplasm, although it still retained the NLS. As with full-length Tax-GFP, the cytoplasmic localization of Tax⌬214-GFP was not altered by treatment of cells with LMB, providing additional support that Tax nuclear export is not directed by CRM-1. These results suggest that the localization of Tax may be directed by specific changes in Tax conformation or by specific interactions with other cellular proteins that could lead to changes in the availability of the Tax NES and NLS.

EXPERIMENTAL PROCEDURES
Cell Culture-HeLa and U-87 MG cells were grown in Dulbecco's modified Eagle's medium. Jurkat cells were grown in RPMI 1640 medium. For each cell line, the growth medium was supplemented with fetal bovine serum (10%), antibiotics (penicillin, streptomycin, and kanamycin at 0.04 mg/ml each), L-glutamine (0.3 mg/ml), and sodium bicarbonate (0.05%). Cell lines were maintained at 37°C in 5% CO 2 at 90% relative humidity.
Fusion Protein Construction and Plasmid DNA Purification-The cDNA coding sequences for Tax and the Tax⌬214 truncation mutant FIG. 1. HTLV-I Tax protein binding and signal domains and Tax-GFP chimeric constructs. A, current known protein-binding domains residing within the HTLV-I Tax protein; B, comparison of the leucine-rich NES of Tax with those of other retroviral proteins; C, Western immunoblot analysis of GFP chimeric constructs used in this study. Cell lysates of transfected HeLa or U-87 MG cells were subjected to electrophoresis on a 10% Tris-HCl/SDS-polyacrylamide gel and then blotted onto nitrocellulose. GFP chimeric constructs were detected using a anti-GFP polyclonal antibody. Arrowheads represent the bands corresponding to the construct names listed at the top of each lane. Blots were stripped of antibody and reprobed with an anti-tubulin polyclonal antibody to control for lane loading differences. CBP, cAMP response element-binding protein-binding protein; CREB, cAMP response element-binding protein; HIV-1, human immunodeficiency virus type 1; SIV, simian immunodeficiency virus; VMV, visna maedi virus.
protein were cloned into pEGFP-N1 (Clontech) using PCR and Taxspecific primers. This resulted in a protein expression construct in which Tax was fused to the amino terminus of GFP. The Tax NES (tNES)-GFP and tNES(L194A)-GFP recombinant plasmids were constructed by cloning double-stranded oligonucleotides into the HindIII and SstII restriction endonuclease sites within the pEGFP-N1 plasmid. Point mutations of the NES within the context of full-length Tax-GFP were constructed using oligonucleotides encoding the mutations and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. Plasmid DNA used for screening and automated sequencing was isolated using the Concert miniprep system (Invitrogen) as described by the manufacturer. The nucleotide sequences of all plasmid constructs were confirmed by automated sequencing (Macromolecular Core Facilities of the Pennsylvania State University College of Medicine) and subsequent bioinformatics analysis using Lasergene software (DNASTAR, Inc., Madison, WI). Western Immunoblot Analyses-After transfection (24 h), the cell culture medium was collected and subjected to centrifugation at 16,000 ϫ g for 1 min. The resultant cell pellet was washed once with cold phosphate-buffered saline (PBS; 1 ml). Cells attached to the culture dish were also washed once with cold PBS (1 ml), scraped off the culture dish using a rubber cell scraper, and then added to the cell pellet. Cells were lysed using the mammalian protein extraction reagent (200 l for cells transfected with GFP and 50 l for the remaining reactions; Pierce) and incubated on ice for 10 min. Cell debris was pelleted by centrifugation at 16,000 ϫ g for 10 min. The lysate (15 l) was added to Laemmli sample buffer (15 l; Bio-Rad) and heated at 95°C for 5 min. Samples were loaded onto a 10% Tris-HCl/SDS-polyacrylamide gel (Bio-Rad), subjected to electrophoresis at 180 V for 45 min, and blotted onto nitrocellulose (Pall Corp., Ann Arbor, MI) for 2 h at 40 mA. Blots were blocked for 15 min with 5% milk dissolved in a solution of PBS with 1.0% Tween 20 (PBST), rinsed once for 10 min and twice for 5 min with PBST, incubated for 1 h with the GFP-specific antibody ab-290 (Abcam Ltd., Cambridge, UK), rinsed once for 10 min and twice for 5 min with PBST, and incubated for 1 h with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (Amersham Biosciences). Finally, blots were washed again once for 10 min and twice for 5 min with PBST, developed using the Western Lightning kit (PerkinElmer Life Sciences), and exposed to x-ray film. Subsequently, blots were stripped of both the primary and secondary antibodies utilizing Restore Western blot stripping buffer (50 ml; Pierce) following the manufacturer's instructions. To control for the amount of protein loaded per well, blots were probed with an anti-tubulin primary antibody (Sigma) and a horseradish peroxidase-conjugated anti-goat secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Nuclear and Cytoplasmic Extraction-HeLa or U-87 MG cells were plated in six-well plates and transfected as described above. After transfection (24 h), the medium was removed, and cells were washed once with cold PBS (1 ml). PBS (0.5 ml) was added to each well, and cells were scraped off using a rubber cell scraper and transferred to a 1.5-ml microcentrifuge tube. Cells were pelleted by centrifugation at 1000 ϫ g for 2 min, and the PBS was removed. Nuclear and cytoplasmic protein fractions were isolated using the nuclear extraction-protein extraction reagent (Pierce). Fractions were subjected to SDS-PAGE and Western immunoblot analyses as described above. The presence of transfected proteins was detected using the anti-GFP primary antibody ab-6663 (Abcam Ltd.) and the horseradish peroxidase-conjugated antirabbit secondary antibody. Protein bands of interest were scanned using an Eastman Kodak Image Station 440CF, and band intensities were quantified using Eastman Kodak 1D Image Analysis software.
Microscopic Analyses-Transfected cells were prepared for microscopic analysis ϳ24 h after transfection. In preparation for fixation of cells, the medium was aspirated, and cells were washed once with 0.2 ml of PBS. Cells were fixed for 30 min with 4% paraformaldehyde, washed twice for 5 min with PBS, permeabilized with 0.5% Triton X-100 in PBS for 15 min, and washed again twice for 5 min with PBS. Actin and nucleic acids were stained for 15 min with phalloidin-Alexa 546 (82.5 nM; Molecular Probes, Inc., Eugene, OR) and for 2 min with 4,6-diamidino-2-phenylindole (100 pM; Molecular Probes, Inc.) diluted in PBS. Coverslips were added along with Fluormount-G mounting medium (Southern Biotechnology Associates, Inc., Birmingham, AL). Cells were visualized using an Olympus IX-81 automated microscope equipped with appropriate filter cubes for visualizing 4,6-diamidino-2phenylindole, Alexa 546, and enhanced GFP (EGFP). Images were obtained with a Cook CCD Sensicam digital camera controlled by Slidebook software (Intelligent Imaging Innovations, Denver, CO). All components of the microscopy system were controlled using an Apple Macintosh G4 dual 1-GHz processor computer. Raw fluorescent images were deconvolved (no-neighbors method) using Slidebook.
For imaging of live cells transfected with yellow fluorescent protein (YFP), Tax-YFP, or Tax⌬214-YFP along with cyan fluorescent protein (CFP)-Nuc (Clontech), no preparation was performed prior to microscopic analysis. For imaging of live cells transfected with GFP constructs, cells were incubated with 1 mM Hoechst 33342 (Molecular Probes, Inc.) diluted in culture medium for 10 min immediately prior to microscopic analysis. Cells were visualized using an Olympus IX-81 automated microscope equipped with appropriate filter cubes for visualizing Hoechst and EGFP or YFP and CFP, and images were obtained as described above.
Quantification of Cytoplasmic Tax-GFP after Microscopic Analysis-HeLa and U-87 MG cells were plated on fibronectin-coated slides, transfected with Tax-GFP in the absence or presence of LMB, and then prepared for microscopic analysis as described above. Images of 50 cells from each treatment group were obtained and deconvolved (no-neighbors method) using Slidebook. For each cell image (see Fig. 2), three masks highlighting Tax-GFP, the nucleus, or the cytoplasm were constructed using either the mask segmentation or mask creation and drawing tools within Slidebook. Mask operations were then utilized to isolate Tax-GFP localized within either the nucleus or cytoplasm. Submasks were constructed to determine the square area (m 2 ) of each Tax-GFP point. The total area of Tax-GFP in each compartment (cytoplasm or nucleus) was calculated and summed. The percent of total Tax-GFP in the cytoplasm was determined by utilizing the formula (c/(c ϩ n)) ϫ 100, where c is the total square area of cytoplasmic Tax-GFP and n is the total square area of nuclear Tax-GFP per cell.
Statistical Analyses-Data collected for the quantitation of cytoplasmic Tax-GFP were analyzed for statistical significance using JMP Version 5.0 (SAS Institute, Cary, NC). Briefly, each set of data was imported into JMP, and an analysis of variance was performed on the four data groups as a whole. The statistical significance of each of the indicated comparisons (see Fig. 3B) was determined using Student's t test.

Tax-YFP/GFP Intracellular Distribution Is
Cell Typedependent-Several studies have examined the intracellular distribution of the HTLV-I Tax protein (14,(27)(28)(29). These investigations have indicated that Tax localizes mainly to the nucleus in interchromatin granules and spliceosomal speckles, areas of the genome where high rates of transcription are thought to occur (30). In these studies, very little Tax was detected in the cytoplasm of the cell types examined, including HeLa and COS-7 cells as well as the HTLV-I-infected cell lines C8166-45 and MT2. However, in more recent studies performed in selected T cell lines, HeLa cells, and primary astrocytes or astrocytic cell lines infected with HTLV-I, Tax was shown to accumulate to significant levels in the cytoplasm as well as the nucleus (24,26,31,32). Cumulatively, these studies have provided the foundation for additional experiments focused on defining the cellular and viral parameters that regulate the nucleocytoplasmic distribution of Tax.
To facilitate studies to examine the intracellular localization of Tax in live cells, the Tax DNA coding sequence was fused to the coding sequence for YFP (pEYFP-N1, Clontech). The intracellular localization of the Tax-YFP chimeric protein was examined in several cell lines in which the nuclear marker CFP-Nuc was also transfected. CD4 ϩ cells serve as a primary HTLV-I target cell during the genesis of both ATL and HAM/ TSP; thus, the Jurkat cell line was selected as a representative CD4 ϩ T cell line for initial experimentation. In this cell line, Tax localized primarily to the nucleus (Fig. 2, upper panels). The nuclear localization of Tax-YFP in Jurkat cells was similar to that described in previous reports of Tax localization in T cells utilizing indirect immunofluorescence (33). In conjunction with these studies, the intracellular localization of Tax-YFP was also examined in HeLa cells based on their extensive use in previous studies that have focused on the intracellular localization of Tax (24,29,30). As previously described utilizing indirect immunofluorescence analyses, transfection of HeLa cells with the Tax-YFP construct resulted in the localization of Tax to the nucleus in a punctate pattern (Fig. 2, middle panels). However, by visual comparison, the amount of cytoplasmic localization of Tax-YFP in HeLa cells occurred to a greater degree than previously reported (Fig. 2, middle panels).
Previous studies have demonstrated that the degree of Tax localized to the cytoplasm differs based on both the cell type used to study localization and the detection system used to identify Tax (31,32). The previous studies cited utilized either immunofluorescence in concert with an anti-Tax antibody (4C5) that recognizes amino acids 333-353 or a fusion of Tax and the fluorescent protein Discosoma sp. red fluorescent protein or EGFP. Antibody 4C5 was found to detect Tax in different nucleocytoplasmic ratios depending on the cell type used for detection. For instance, antibody 4C5 was reported to detect Tax in the nucleus of JPX cells (a Jurkat-derived cell line that stably expresses Tax), in the nucleus and cytoplasm of PX1 cells (fibroblasts isolated from a Tax transgenic mouse), and predominantly in the cytoplasm of passage 3 primary T cells cultured from peripheral blood lymphocytes of ATL patients (31). In addition, Tax-Discosoma sp. red fluorescent protein and Tax-EGFP were both found to localize to the nucleus and cytoplasm in "hot spots" when transiently transfected into either human osteosarcoma or H441 cells (32). Both of these studies utilized methods similar to the methods presented here in that only full-length Tax or an isoform of Tax in which the carboxyl terminus was available for antibody binding was detected. Previous studies of Tax intracellular localization in HeLa cells that demonstrated strong nuclear localization and very little cytoplasmic localization may have utilized antibodies directed against other regions of Tax that were not available when Tax was localized in the cytoplasm (24,30). Finally, it is possible that the YFP tag utilized in the studies presented herein promoted Tax nuclear export or inhibited Tax nuclear import, resulting in increased levels of cytoplasmic localization. However, we consider this to be an unlikely event based on the ubiquitous utilization of YFP in many other studies of protein intracellular localization in which the YFP tag did not appear to alter the course of cellular localization of the native protein.
Furthermore, the degree of cytoplasmic localization of Tax-GFP in HeLa cells was also observed to be different between cells within the same population (data not shown). This could be the result of a number of factors, including, but not limited to, stage of the cell cycle and differential expression levels of protein(s) involved in Tax cytoplasmic localization.
Because cervical epithelial cells are not likely infected by HTLV-I during the course of either ATL or HAM/TSP, the intracellular localization of Tax-YFP was also examined in the astrocytic U-87 MG cell line. Although in vivo results have not shown conclusively that astrocytes are infected in HTLV-Ipositive individuals, experimental results utilizing primary astrocytes infected with HTLV-I in vitro have suggested that astrocytes may be infected in vivo and that viral infection of this cell type may play a role in the pathogenesis of HAM/TSP (25,26,34,35). In previous studies, HTLV-I-infected astrocytes exhibited Tax accumulation in the nucleus and also to a significant extent in the cytoplasm (26). In agreement with previous studies, we have also demonstrated that Tax-YFP was capable of localizing to both the nucleus and cytoplasm of live U-87 MG cells (Fig. 2). The differential localization of Tax-YFP between the cell line models of two pathogenically relevant cell types examined in this study (the Jurkat T and astrocytic U-87 MG cell lines) suggested the possibility that differential intracellular localization of Tax within different HTLV-I-susceptible cell populations or within the same cell population under different physiological conditions may alter the level of interactions of Tax with selected cellular pathways such as the checkpoint control and NF-B signaling pathways. The phenotypic display of Tax localized to the cytoplasm in punctate spots in both previous studies and the studies presented herein is supportive of an interaction of Tax with cellular cytoplasmic proteins. This localization may be the result of Tax interaction with specific proteins located within the cytoplasm or within cytoplasmic organelles such as the endoplasmic reticulum and Golgi complex because Tax has been suggested to interface with pathways involved in exocytosis (36).
Tax as a signal to direct protein transport out of the nucleus (Fig.  1C) (15,16). As previously discussed (24), examination of the Tax protein sequence reveals a putative leucine/isoleucine-rich NES between amino acids 188 and 202. This sequence is also similar to other characterized retroviral NESs, including the NES in the human immunodeficiency virus type 1 protein Rev (Fig. 1B). Most studies pertaining to nuclear export have implicated the CRM-1 protein in facilitating the export of NEScontaining proteins. Conveniently, an antibiotic compound, LMB, has been found to specifically inhibit CRM-1-mediated nuclear export (37)(38)(39). Based on the identification of a putative NES in Tax by sequence analysis, we proceeded to determine whether the addition of LMB would inhibit the cytoplasmic localization of Tax. To effectively distinguish between the cytoplasm of adjacent cells, we also stained for cytoplasmic components. This required the fixation of the cells before staining with phalloidin, a stain specific for the cellular cytoskeleton. However, it was first determined whether fixation of cells altered the nucleocytoplasmic localization of Tax. In this regard, two assays were performed. First, a cell fractionation Cells were fixed and stained as described under "Experimental Procedures." A, images of cells representative of the entire population are shown. Cytoplasmic actin (phalloidin) is shown in red, the nucleus (4,6-diamidino-2-phenylindole) in blue, and Tax-GFP in green. Cells were viewed on an inverted fluorescence microscope using a ϫ40 objective as described under "Experimental Procedures." After image capture, each image was deconvolved using the no-neighbors method within Slidebook. B, the amount of cytoplasmic Tax-GFP in a population of cells (n ϭ 50) for each experiment is represented as the percent of the total amount of Tax-GFP per cell that was localized to the cytoplasm. LMB treatment did not affect the percent of Tax-GFP in the cytoplasm in either cell type. Also, U-87 MG cells, with or without LMB treatment, contained a significantly (*, p Ͻ 0.01) higher percent of cytoplasmic Tax-GFP than HeLa cells with or without LMB treatment. C, shown are the results of a nuclear (N) and cytoplasmic (C) fractionation experiment carried out with YFP-or Tax-YFP-transfected HeLa and U-87 MG cells treated with or without LMB (40 M) for 3 h. analysis was performed in which the relative levels of Tax-YFP present in the nuclear and cytoplasmic fractions were determined. Second, a microscopic analysis was performed in which the percent of Tax-YFP in the cytoplasm of live cells was determined. The results using the nuclear and cytoplasmic fractionation analysis indicated that 69.6 and 57.4% of Tax were localized to the cytoplasm in U-87 MG and HeLa cells, respectively (a difference of 1.3-fold) (Fig. 3C). Additionally, the microscopic analysis with live cells indicated that the percent of Tax per cell localized to the cytoplasm was also greater for U-87 MG cells (57.6%) than for HeLa cells (29.2%; a difference of 2.0-fold) (data not shown). Both of these assays confirmed Tax localization to the cytoplasm in U-87 MG and HeLa cells. With these results in hand, Tax-GFP-transfected cells were fixed with paraformaldehyde, and the average amount of cytoplas-mic Tax-GFP per cell type was determined by microscopic analysis (Fig. 3B). This analysis indicated that the percent of cytoplasmic Tax in U-87 MG cells was 81.0%, whereas that in HeLa cells was 49.0%, a difference of 1.7-fold. With each analysis performed, there were large differences in the amount of cytoplasmic Tax between cells of the same culture. This point was demonstrated by the amount of S.D. in the fixed-cell microscopic analysis (Fig. 3B). The high level of variability exhibited by these results may very well be due to differences between live and fixed cells or simply due to the innate amount of variation that would be observed in experimentation. In any case, the primary purpose for these analyses was to determine a benchmark amount of cytoplasmic Tax to assess whether LMB had any effect on the nucleocytoplasmic localization of Tax. Subsequently, HeLa and U-87 MG cells transfected with Tax-GFP were exposed for 3 h to LMB (40 M). However, contrary to our expectations, Tax-GFP intracellular distribution was not altered in the presence of LMB in either HeLa or U-87 MG cells (Fig. 3A). This observation was confirmed by microscopic determination of the average amount of cytoplasmic Tax (Fig. 3B). For each cell type, the percent of Tax-GFP localized to the cytoplasm in LMB-treated cells was not significantly different from that in untreated cells. It is possible that the Tax NES effectively competed with LMB for binding to CRM-1, and thus, a higher concentration of LMB was necessary to visualize inhibition of nuclear export. Consequently, the localization of Tax-GFP was assessed using increasing concentrations of LMB (40, 80, and 120 M) over a 3-h period. Even at the highest concentration examined, Tax-GFP localization was similar to that in untreated cells (data not shown). These results were confirmed by performing nuclear and cytoplasmic extractions of Tax-YFP-transfected cells in the absence or presence of LMB (40 M) for 3 h. In agreement with the microscopic analyses, the amount of cytoplasmic and nuclear Tax did not change upon LMB treatment (Fig. 3C). These results suggest that Tax nuclear export is facilitated by an LMB-insensitive process and is therefore independent of CRM-1.
The Tax NES Functions to Direct Nuclear Export of GFP in an LMB-sensitive Manner-To further examine the functionality of the Tax NES, the DNA sequence for amino acids 188 -202 of Tax was fused to the carboxyl terminus of GFP (pEGFP-C3, Clontech), yielding tNES-GFP (Fig. 1B). This construct was then transfected into both HeLa and U-87 MG cells. Although wild-type GFP localized almost equally to both the nucleus and cytoplasm (Fig. 4A), tNES-GFP localized almost exclusively to the cytoplasm in both cell types (Fig. 4B). This result strongly suggests that the Tax NES is functional and directs the export of proteins out of the nucleus. To determine whether the tNES-GFP protein was effectively shuttling between the nucleus and cytoplasm via the CRM-1 pathway, treatment of transfected cells with LMB was again employed. After a 3-h incubation with LMB (40 M), the tNES-GFP construct was localized to both the nucleus and cytoplasm (Fig. 4B). Inhibition of nuclear export was detected as early as 1 h after the addition of LMB (data not shown). A tNES-GFP construct was also designed in which Leu 194 was mutated to Ala (tNES(L194A)-GFP) (Fig.  1B). Previous reports have determined that mutation of key leucine or isoleucine residues within an NES abrogates CRM-1-mediated nuclear export. As predicted, localization of tNES(L194A)-GFP was shown to be similar to that of GFP alone and was also LMB-insensitive (Fig. 4C). During this experiment, protein synthesis continued to occur; thus, newly made tNES-GFP remained in the cytoplasm after synthesis, explaining the presence of cytoplasmic GFP after the addition of LMB. Both tNES-GFP and tNES(L194A)-GFP were shown to be expressed in HeLa and U-87 MG cells by Western immunoblot analyses (Fig. 1C). These results demonstrate that, unlike that of full-length Tax-GFP with the NES in its native orientation, direction of nuclear export by the Tax NES within the context of another protein and likely exposed to the export pathway can direct protein trafficking utilizing the CRM-1 nuclear export pathway.
Tax(L200A)-GFP Nuclear Export Is Inhibited-Although the results presented have shown that the Tax NES is a functional NES in the context of the tNES-GFP chimera, it was necessary to examine the function of the NES within the context of the full-length Tax protein. To do this, point mutant Tax-GFP constructs were designed in which the coding sequence for each critical leucine or isoleucine residue within the NES was mutated to an alanine coding sequence. The intracellular localization of these proteins was then examined in living cultures of HeLa and U-87 MG cells. Four of the mutants exhibited no major changes with respect to their cytoplasmic localization. However, the localization of the Tax(L200A)-GFP mutant protein in U-87 MG cells was clearly different from that of the wild-type protein (Fig. 5). Specifically, this mutant localized strongly to the nuclear membrane as well as diffusely throughout the nucleus in the majority of transfected U-87 MG cells. This was most likely due to a defect in shuttling of Tax or binding of Tax with the cellular shuttling protein(s) responsible for Tax nuclear export. As with wild-type Tax-GFP, this mutant still localized to interchromatin granules and spliceosomal speckles, indicating that it was still able to bind host transcriptional machinery. There was still some cytoplasmic accumulation of Tax(L200A)-GFP. This was likely newly translated Tax that had not yet trafficked to the nucleus. Additionally, previous studies have suggested that Tax is secreted from cells (40); thus, this could also be Tax localized to cellular organelles associated with the secretory process.
Tax Contains a Masked NES-Although experimental results have indicated that the Tax NES can function to direct GFP nuclear export, the fact that native full-length Tax localizes to both the nucleus and cytoplasm suggests that nuclear export of Tax is a regulated event. This regulation may occur due to altered interactions between Tax and cellular proteins, resulting in the exposure and recognition of the Tax NES by the cellular nuclear export machinery. Recently, it has been reported that nuclear export of the NES-containing and resident nuclear protein INI1/nSNF5 may be regulated in such a manner (41). In these studies, the carboxyl terminus of INI1/nSNF5 was truncated to the NES, in theory exposing the NES to the cellular export machinery. As hypothesized, this construct localized almost exclusively to the cytoplasm, suggesting that exposure of the normally masked NES to the cellular export machinery is necessary for INI1/nSNF5 nuclear export. Based on these results, a study was designed to determine whether Tax also contains a masked NES. To this end, Tax was truncated at amino acid 214 (Tax⌬214-GFP), removing the carboxyl-terminal section of the protein shortly after the NES (Fig.  1B). As determined by Western immunoblot analysis (Fig. 1C), this construct was expressed in both HeLa and U-87 MG cells. There was a significant change in the localization of Tax⌬214-GFP (Fig. 6) in HeLa and U-87 MG cells. Specifically, Tax⌬214-GFP localized primarily to the cytoplasm in both cell lines, even though the truncated protein still contained the NLS. This result was not obtained based on prevention of nuclear import due to an altered tertiary structure that abrogates the NLS because Tax⌬214-GFP in several cells was still able to enter the nucleus (Fig. 6). The nuclear export of Tax⌬214-GFP was also not inhibited by LMB (40 M, 3 h), as concluded from both microscopic (Fig. 6A) as well as nuclear and cytoplasmic fractionation ( Fig. 6B) analyses, again suggesting that Tax nuclear export is not directed by CRM-1. Additionally, the distribution pattern of Tax⌬214-GFP within the cytoplasm was more diffuse than that observed for Tax-GFP. This result indicates that the Tax carboxyl terminus is important with respect to the interaction of Tax with cytoplasmic proteins or may contain amino acid sequences important in the specific localization of Tax to discrete cytoplasmic compartments. DISCUSSION Because of the small size of retroviral genomes, retroviral gene products must perform multiple functions essential to both viral replication and pathogenesis. Consistent with this view, Tax is involved in several cellular signaling and transcriptional control pathways based on interactions with numerous cellular proteins. In most cell types, Tax accumulates primarily in the nucleus, an indication of the important role it plays as a transcriptional transactivator. However, for Tax to interact with the NF-B pathway, it must also localize to the cytoplasm. Cytoplasmic localization of Tax could result from the lack of NLS recognition and nuclear import shortly after protein synthesis or exit from the nucleus via a cellular nuclear export system. The latter is more likely because supportive evidence for nuclear export and cytoplasmic accumulation was first reported when it was shown that Tax could shuttle be-tween the nuclei of two heterokaryon cells (24). This is not an unusual functional property for proteins involved in transcriptional regulation because several reports have demonstrated that differential localization of proteins can function to either inhibit or induce a cellular process (23,42). Relevant to this process, Tax interaction with MAD1 and the resulting loss of cell checkpoint control and the appearance of multinucleated giant cells may be the result of the nuclear export capabilities of Tax. Additionally, previous studies (14,(27)(28)(29)31) of Tax intracellular localization utilizing several cell types and methods of Tax detection have shown that the amount of cytoplasmic Tax can differ significantly. For example, Tax was detected specifically in the nucleus of JPX cells, a Jurkat-derived cell line that stably expresses Tax, and primarily in the cytoplasm of T cells derived from ATL patients (31). Cell type differences such as these may account for differential efficiencies of Tax transactivation, viral replication, and viral pathogenesis between specific cell populations.
The results presented in this report begin to unravel the mechanism of Tax nucleocytoplasmic shuttling and its possible role in the pathogenesis of HTLV-I. Although reports of the discovery of nuclear export sequences have become almost commonplace, the role that nuclear export plays in protein function and regulation can differ significantly between proteins. Although we have determined that the Tax NES itself can direct nuclear export of GFP via the CRM-1 pathway, it is interesting that it does not direct full-length Tax nuclear export utilizing this system (Fig. 7). At first, this could be attributed to a dysfunction in the Tax NES; however, this is unlikely because the Tax(L200A)-GFP mutant localized distinctively to the nuclear membrane in the majority of U-87 MG cells transfected with this construct. Additionally, the NES within Tax must be important in Tax function because nonfunctional elements within a retroviral protein could be expected to be lost as the result of a lack of selective pressure for their retention. Several HTLV-I phylogenetic studies corroborate this theory because none of the HTLV-I genomes sequenced to date contain amino acid alterations within the NES (43)(44)(45)(46)(47). Additionally, although the CRM-1 nuclear export pathway has been the most extensively studied to date, it is possible that other nuclear export pathways exist that recognize the same leucine-rich sequence. In support of this, it has been recently reported that calreticulin, a calcium-binding protein, is also able to function as a nuclear export-directing protein (48,49). Calreticulin effectively interacts with the leucine-rich sequence on protein FIG. 7. Model of Tax nuclear export. Tax nuclear export may occur through one of three proposed pathways, all involving a nuclear export protein. The first pathway assumes that the Tax NES is obscured by a protein-protein interaction within the nucleus and is uncovered only after that interaction has been terminated. The second pathway assumes that the Tax NES is masked and is exposed only upon interaction with a secondary protein. The third pathway assumes that a secondary protein modification of Tax (phosphorylation, acetylation, etc.) is required for Tax NES interaction with the nuclear export pathway. Our results suggest that Tax nuclear export does not occur via the CRM-1 pathway; however, Tax nuclear export may occur from an interaction between Tax and calreticulin or another as yet uncharacterized NES-recognizing nuclear export protein.
kinase inhibitor in a Ran GTPase-dependent manner and can transport protein kinase inhibitor out of the nucleus and into the cytosol in an LMB-insensitive manner. Thus, because these results are similar to the results presented herein, future studies of Tax nuclear export will involve investigations concerning the interaction of Tax with calreticulin. This may be one of many nuclear export pathways that interface with Tax to facilitate nucleocytoplasmic shuttling.
We have also reported that the Tax NES may function as a masked NES, much in the same manner as the INI1/nSNF5 protein (41). By truncating and removing the carboxyl terminus of Tax at amino acid 214, constitutive activation of nuclear export was achieved. This phenotype could have been the result of a blocked NLS resulting from an altered conformation of the truncated protein. However, several cells in the localization studies of Tax⌬214-GFP contained protein within the nucleus, strongly suggesting that this fusion protein was able to enter the nucleus (Fig. 5). Thus, the localization of Tax⌬214-GFP provided evidence that nuclear export of Tax may be regulated and dependent on several factors (Fig. 6). First, the Tax NES may be hidden by a protein-protein interaction, allowing the Tax NLS to remain the predominant localization signal and directing protein localization to the nucleus. Second, Tax tertiary structure may be altered during the succession of steps in which the NES is subsequently exposed to cellular export machinery. Alternatively, Tax nuclear export may be regulated through a secondary protein modification such as phosphorylation or acetylation. In this regard, Tax phosphorylation has been studied in several cell types, and alterations in the pattern of Tax accumulation within the cytoplasm have not been reported (50,51).
HeLa cells have been commonly used to study Tax intracellular localization; and as a result, we have included them in this study for comparison with other reports on Tax intracellular localization. Astrocytic U-87 MG cells represent a cell line with both pathogenic relevancy and a significant amount of cytoplasmic Tax. It has previously been reported that Tax can localize to the cytoplasm to a significant extent in both an HTLV-I-infected astrocytic cell line and primary astrocytes (26). Although there is still a debate as to whether astrocytes are infected in HTLV-I-positive individuals, there is no debate as to the importance of the astrocyte with respect to normal central nervous system function and the pathogenic consequences if astrocytic regulation is disrupted. The role of Tax in astrocytic dysfunction has been demonstrated and has been shown to involve Tax-induced up-regulation and release of tumor necrosis factor-␣, matrix metalloproteinase-2, and matrix metalloproteinase-9. Importantly, the cytoplasmic localization of Tax is crucial to the up-regulation of tumor necrosis factor-␣ and matrix metalloproteinase-9, as the transcriptional activation of both proteins occurs via the NF-B pathway (42,52,53). Continued analyses need to be performed to address the specific issues of Tax nucleocytoplasmic distribution and its role in the pathogenesis of HTLV-I.