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Interaction of HTLV-1 Tax protein with calreticulin: Implications for Tax nuclear export and secretion
Biomedicine & Pharmacotherapy 61 (2007) 194e200 www.elsevier.com/locate/biopha
Original article
Interaction of HTLV-1 Tax protein with calreticulin: Implications for Tax nuclear export and secretion Timothy Alefantis a, Katherine E. Flaig b, Brian Wigdahl b, Pooja Jain b,* b
a Vital Probes, Inc., 1300 Old Plank Road, Mayfield, PA 18433, USA Department of Microbiology and Immunology, and Center for Molecular Virology and Neuroimmunology, Center for Cancer Biology, Institute of Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA 19102, USA
Received 25 January 2007; accepted 6 February 2007 Available online 9 March 2007
Abstract Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP). The HTLV-1 transcriptional transactivator protein Tax plays an integral role in virus replication and disease progression. Traditionally, Tax is described as a nuclear protein where it performs its primary role as a transcriptional transactivator. However, recent studies have clearly shown that Tax can also be localized to the cytoplasm where it has been shown to interact with a number of host transcription factors most notably NF-kB, constitutive expression of which is directly related to the T cell transforming properties of Tax in ATL patients. The presence of a functional nuclear export signal (NES) within Tax and the secretion of full-length Tax have also been demonstrated previously. Additionally, release of Tax from HTLV-1-infected cells and the presence of cell-free Tax was demonstrated in the CSF of HAM/TSP patients suggesting that the progression to HAM/TSP might be mediated by the ability of Tax to function as an extracellular cytokine. Therefore, in both ATL and HAM/TSP Tax nuclear export and nucleocytoplasmic shuttling may play a critical role, the mechanism of which remains unknown. In this study, we have demonstrated that the calcium binding protein calreticulin interacts with Tax by co-immunoprecipitation. This interaction was found to localize to a region at or near the nuclear membrane. In addition, differential expression of calreticulin was demonstrated in various cell types that correlated with their ability to retain cytoplasmic Tax, particularly in astrocytes. Finally, a comparison of a number of HTLV-1-infected T cell lines to non-infected T cells revealed higher expression of calreticulin in infected cells implicating a direct role for this protein in HTLV-1 infection. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: HTLV-1 Tax; Nucleocytoplasmic shuttling; Calreticulin
1. Introduction HTLV-1 is the etiologic agent of two major diseases; a progressive lymphoma designated adult T cell leukemia (ATL) and a debilitating neurological disease known as
* Corresponding author. Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, 18 th floor, New College Building, Room #18311, Drexel University College of Medicine, 245 North 15 th Street, Philadelphia, PA 19102, USA. Tel.: þ1 215 762 8586; fax: þ1 215 762 1955. E-mail address: [email protected] (P. Jain). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.02.005
HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 oncoprotein Tax has been studied extensively with respect to its role in regulating cellular and viral gene expression during the course of HTLV-1 infection [1e3]. Specifically, within the context of the viral life cycle, Tax interacts with host transcriptional machinery, especially the ATF/ CREB pathway, to facilitate the binding of selected cellular transcription factors to the viral long terminal repeat (LTR) within the nucleus. In addition to the ATF/CREB pathway, Tax also interacts with the NF-kB signaling pathway in the cytoplasm to induce NF-kB translocation to the nucleus by binding to IKKg, a subunit of the IKK complex [4e7]. The ability of Tax to induce
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the NF-kB pathway has been intensely studied because of its possible role in cellular transformation and the onset of ATL in HTLV-1-infected individuals [4e6,8]. It is unknown whether Tax that interacts with NF-kB in the cytoplasm represents newly synthesized protein resident in the cytoplasm, or whether it represents protein shuttled from the nucleus back to the cytoplasm. If it is the latter, or a combination of the two, the regulation of Tax nucleocytoplasmic shuttling is important since the differential availability of Tax to the NF-kB as compared to ATF/CREB pathway could be expected to have different effects on cellular metabolism and progression of HTLV-1-associated pathogenesis. In addition to its intracellular functions, Tax is also known to cause a variety of effects on cells as an extracellular protein particularly in the context of HAM/TSP. The presence of antibodies against Tax is demonstrated in a majority of HTLV-1-infected individuals [9,10]. Recently, cell-free Tax has been detected in the CSF of HAM/TSP patients [11]. These observations, along with a number of studies including our own, demonstrating the effects of extracellular Tax on both lymphoid and non-lymphoid cells, suggest that Tax is available for immune recognition and can function as an extracellular cytokine [12e 21]. However, it is not yet certain whether the cell-free Tax was the result of apoptosis or necrosis of HTLV-1-infected cells or if it was secreted from the infected cell populations. Consistent with the concept of cellular secretion, the presence of a leucine-rich NES between amino acids 188e200 of Tax has been reported [22]. The secretion of Tax has also been reported from HTLV-1-infected and Tax-transfected cells [16,23]. At any given time, an HTLV-1-infected cell may contain a rate-limiting amount of Tax. Thus, an alteration in the balance of Tax nucleocytoplasmic shuttling may have functional impact on the Tax-induced signaling pathways in both the nucleus and cytoplasm. Furthermore, we hypothesize that Tax may localize to the cytoplasm as an intermediary step during the course of its release from HTLV-1-infected cells, as demonstrated by the presence of Tax in cytoplasmic secretory-like vesicles [23]. Therefore, understanding the mechanism(s) of Tax nuclear export and nucleocytoplasmic shuttling will be critical in clarifying the overall processes associated with HTLV-1-associated immune and central nervous system disease. Traditionally, nuclear export of proteins containing a leucine-rich NES has been shown to be mediated by CRM-1 nuclear export receptor via the nuclear pore complex [24e26]. Previous observations have indicated that the Tax NES (tNES) was shown to function in a CRM-1-dependent manner when fused as an isolated element directly to green fluorescent protein (GFP), the same was not true when the tNES was within the context of full-length Tax [22]. The nucleocytoplasmic localization of full-length Tax-GFP was not altered following treatment of transfected cells with leptomycin B (LMB), an inhibitor of CRM-1 nuclear export. Similarly, studies investigating the nuclear export of a leucine-rich NES containing protein, protein kinase inhibitor (PKI), demonstrated that in a system with CRM-1-depleted cytosol, nuclear export of PKI was retained [27]. These studies have strongly suggested that there are additional export receptors present in mammalian cells that can mediate a CRM-1-independent mechanism
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for nuclear export. Recently the calcium-binding protein implicated in protein folding in the ER lumen, calreticulin, was demonstrated to act as a nuclear export protein [25,27,28]. Calreticulin was demonstrated to facilitate the nuclear export of PKI by a pathway independent of CRM-1 [25]. Several investigators have reported the presence of calreticulin in the nucleus and the cytoplasm [29,30]. Both CRM-1 and calreticulin bind to transport substrates that contain an NES, and require export receptor-substrate-RanGTP complex for export [25]. It has been shown that like HTLV-1 Tax, nuclear export of PKI was not inhibited by LMB. The results presented herein begin to examine the role of calreticulin in Tax nuclear export. Specifically, this investigation demonstrated that calreticulin and Tax interact and co-localize at or near the nuclear membrane in baby hamster kidney (BHK-21) cells, indicating nuclear transport of Tax. Additionally, four HTLV-1-infected T lymphocyte cell lines were demonstrated to contain high levels of calreticulin expression as compared to non-infected Jurkat T lymphocytes. The difference between cells with high levels of cytoplasmic Tax versus those with low levels may be explained by the expression level or relative availability of calreticulin. Therefore, it has been hypothesized that the balance of Tax import and calreticulindependent export from the nucleus provides the cell with a mechanism that may play an important role in regulating Tax biological activity and HTLV-1 induced disease progression. 2. Materials and methods 2.1. Cell culture 293T and BHK-21 cells were grown in Dulbecco‘s modified Eagle’s media. U-87 MG and HeLa cells were grown in Eagle’s minimum essential medium. U-937, Jurkat, and HTLV1-infected T cell lines MT-2, C8166, HuT 102, and SLB-1 were grown in RPMI 1640. Growth media for each cell line was supplemented with 10% fetal bovine serum, 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% CO2 at 90% relative humidity. 2.2. Fusion protein construction and plasmid DNA purification Double-stranded oligonucleotides encoding a six-histidine tag was inserted into the carboxy-terminus of the Tax coding sequence using the QuickChange site-directed mutagenesis kit as described by the manufacturer (Stratagene, La Jolla, CA). The cDNA coding sequence of calreticulin (kindly provided by Dr. Bryce Paschal, University of Virginia School of Medicine) was cloned into the 3xFLAG vector (Sigma, St. Louis, MO) using PCR and calreticulin-specific primers (FLAG-CRT). HTLV-1 Tax constructs for fluorescence microscopy were constructed by cloning full-length Tax cDNA coding sequence into a green fluorescent protein construct
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(pEGFP-N1; Clontech, Palo Alto, CA) yielding a construct encoding fusion proteins in which GFP was fused to the carboxyterminus of Tax as described [22]. Plasmid DNA used for screening and automated sequencing was isolated using the Concert miniprep system as described by the manufacturer (Invitrogen, Carlsbad, CA). The nucleotide sequence of all plasmid constructs was confirmed by automated sequencing (DNA Sequencing Laboratory of the Center for Molecular and Functional Genomics in the Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine) and subsequent bioinformatics analysis using Lasergene software (DNASTAR, Madison, WI). Plasmid DNA used for transient transfections was isolated using the HiSpeed Plasmid Midi kit as described by the manufacturer (Qiagen, Valencia, CA). 2.3. Transient transfections For immunoprecipitation studies, 293T cells were plated in 6-well plates at 1 106 cells per well 24 h prior to transfection. Transfections were performed using 2.4 mg of DNA and Fugene6 transfection reagent (Roche Diagnostics, Mannheim, Germany) with an optimized protocol as described by the manufacturer. For microscopic analyses, BHK-21 cells were plated in an 8-well chamber slide coated with collagen (BD Labware, Bedford, MA) at a concentration of 5 104 cells per well 24 h prior to transfection. Transfections were performed using Fugene6 transfection reagent as described by the manufacturer. 2.4. Immunoprecipitation After transfection (24 h), media was removed and cells were washed once with cold PBS (1 ml). Cells were then lysed and the cell lysate was harvested using the Sigma FLAG immunoprecipitation kit (Sigma) as described by the manufacturer. Anti-FLAG agarose beads were then added to each lysate and the immunoprecipitation was performed as described by the manufacturer (Sigma). After immunoprecipitation (2 h), anti-FLAG agarose beads were washed three times with wash buffer (Sigma), the protein of interest was eluted, and analyzed by western immunoblot analyses (described below) using anti-His6 antibody (ab-9180, 1:6000 dilution, Abcam Ltd, UK).
were then added along with Fluormount-G mounting media (Southern Biotechnology Associates, Inc, Birmingham, AL). Cells were visualized using an Olympus IX-81 automated microscope equipped with the appropriate filter cubes for visualizing Cy-3, and 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-1Ghz processor computer (Apple, Cupertino, CA). Raw fluorescent images were deconvolved (nearest-neighbors method) using Slidebook. 2.6. Whole cell extract preparation Whole cell extracts from Jurkat, 293T, U-87 MG, HeLa, U937 and a number of HTLV-1-infected T cell lines (MT-2, C8166, HuT 102, and SLB-1) were prepared using RIPA buffer [NaCl (150 mM), Igepal (1.0%), deoxycholate (0.5%), Tris pH 8.0 (50 mM)]. Cell debris was cleared from the lysate by centrifugation (5 min, 16,000 g). The protein concentration for each whole cell extract was determined using the DC protein assay as described by the manufacturer (Bio-Rad Laboratories, Hercules, CA). 2.7. Western immunoblot analyses Cell lysates from various cell lines were resuspended in SDS loading buffer and heated at 95 C for 5 min. After heating, samples were loaded onto a 10% TriseHCl SDS polyacrylamide gel (Bio-Rad) and subjected to electrophoresis (180 V, 45 min). Samples were then blotted onto nitrocellulose membrane (Pall Corporation, Ann Arbor, MI) for 2 h (40 mA). Blots were blocked with 5% milk dissolved in a solution of phosphate-buffered saline with 1.0% Tween-20 (PBST, 15 min) and then rinsed with PBST (once for 10 min and twice for 5 min). Blots were incubated with anti-calreticulin antibody (NB 600-101B1, 1:2000 dilution, Novus Biologicals, Littleton, CO), for 1 h at room temperature, rinsed with PBST (once for 10 min and twice for 5 min), and incubated (1 h) with an anti-rabbit horseradish peroxidase-conjugated (HRP) secondary antibody (1:2000 dilution, Amersham Pharmacia, UK). Finally, blots were washed again with PBST (once for 10 min and twice for 5 min) and developed using the Western Lightning kit (Perkin-Elmer Life Sciences, Boston, MA) and exposed to X-ray film.
2.5. Microscopic analyses 3. Results Transfected cells were prepared for microscopy approximately 24 h post-transfection. Cells were fixed for 30 min with paraformaldehyde (4.0%) and washed twice with PBS for 5 min. Cells were then permeabilized with Triton-X 100 (0.5%) in PBS for 15 min and again washed twice for 5 min with PBS. Cells were blocked for 30 min with PBS-BSA (3.0%). FLAG epitope constructs were immunostained using an anti-FLAG-Cy3 antibody (1:1000 dilution in BSA-PBS, 1 h, Sigma, St. Louis, MO). After incubation with antibody, cells were washed three times for 5 min with PBS. Cover slips
3.1. Tax and calreticulin interaction detected by immunoprecipitation Since calreticulin was previously determined to facilitate nuclear export of a leucine-rich NES-containing protein, it was of interest to determine if it could also facilitate the nuclear export of Tax [22,25,31]. Therefore, we have analyzed the interaction of calreticulin and Tax within the context of live cells. An immunoprecipitation assay using 293T cells
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transfected with plasmid constructs encoding either Tax-His6 or FLAG-CRT or a combination of both was utilized. After transfection (24 h), the cell lystes were harvested and antiFLAG agarose beads were utilized to immunoprecipitate FLAG-CRT. Co-immunoprecipitation of Tax-His6 was then detected by western immunoblot analyses using an anti-His6 antibody. As shown in Fig. 1, a band of the predicted 42 kD size of Tax was evident where cells overexpressed both Tax and calreticulin. The negative control samples containing cells transfected with only Tax-His6 or FLAG-CRT did not show a His6-positive band, indicating that the observed immunoprecipitated band was the result of Tax and calrecticulin interaction in intact cells (Fig. 1).
3.2. Tax co-localizes with calreticulin to the nuclear membrane or regions of close proximity Levels of intracellular calreticulin localization differ between cellular compartments due to its multiple functions. While most calrecticulin has been shown to localize to the endoplasmic reticulum based on its role as a molecular chaperone, it has also been shown to accumulate in areas such as the nucleus and secretory pathway. To detail the interaction of Tax with calrecticulin, it was necessary to examine the localization of Tax and calrecticulin within cells. To this end,
Fig. 1. Co-immunoprecipitation of Tax and calreticulin. Cell lysates of TaxHis6-transfected 293T cells were subjected to immunoprecipitation using anti-FLAG agarose beads. Subsequently, co-immunoprecipitated proteins were subjected to electrophoresis on a 10% TriseHCl SDS polyacrylamide gel and then blotted onto nitrocellulose membrane. His6-tag-containing proteins were detected using a His6 polyclonal antibody. The band located at 42 kD represents Tax-His6 co-immunoprecipitated with FLAG-CRT.
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a model cell line BHK-21 (ideal for imaging and localization studies) was co-transfected with Tax-GFP and FLAG-CRT. After 24 h, the cells were fixed and stained with an antiFLAG-Cy3 antibody. A representative image of a Tax-GFP and FLAG-CRT co-transfected BHK-21 that was deconvolved utilizing the nearest neighbors method is shown in Fig. 2. In this image, both Tax-GFP and FLAG-CRT are localized throughout the cell, but are concentrated in an area around the nuclear membrane. Co-localization of these proteins was determined utilizing overlapping masks constructed for both Tax-GFP and FLAG-CRT. Tax-GFP and FLAG-CRT co-localized to an area around the nucleus consistent with the localization at the nuclear membrane and possibly to the nuclear pores, a position that may facilitate Tax egress from the nucleus. These results provide evidence suggesting that Tax interacts with calrecticulin at the nuclear membrane during nucleocytoplasmic transport. 3.3. Endogenous expression of calreticulin differs among various cell types The nucleocytoplasmic distribution of Tax in Jurkat and other T cell lines has been demonstrated in a recent report by Azran et al. [32]. However, cellular distribution of Tax may be different when compared between different cell types, or within specific cell populations at various stages of the cell cycle. For example, both Tax-transfected and HTLV-1-infected astrocytes have been shown to contain a substantial amount of Tax within the cytoplasm [33]. The amount of cytoplasmic Tax can also differ significantly based on the method of Tax expression. For example, Tax was detected specifically in the nucleus of JPX cells (a Jurkat-derived T cell line that stably expresses Tax) and primarily in the cytoplasm of T cells derived from PBMCs of an ATL patient [33]. Cell type differences such as these may account for differential efficiencies of Tax transactivation, viral replication, and overall HTLV-1 pathogenesis. We hypothesized that if calrecticulin is a critical mediator of Tax nuclear export, its endogenous expression in various cell types could account for observed differences in cytoplasmic Tax in various cell types. Therefore, calreticulin expression was examined in various cell types including T cell line (Jurkat), astrocytes (U-87 MG), epithelial cells (293T and HeLa), and monocytes (U-937) by western immunoblot analysis (Fig. 3). It was interesting to note that epithelial cells 293T and HeLa, and astrocytic cell line (U-87 MG) demonstrated higher expression levels of calreticulin as compared to T cells and monocytes for the same amount of total protein used in the analysis. In agreement with previous studies, these results suggest a correlation between higher levels of calreticulin in cell types shown to contain higher levels of cytoplasmic Tax such as astrocytes [33]. 3.4. Higher expression of calreticulin in HTLV-1-infected T cells compared to non-infected cells To correlate these observations with HTLV-1 infection and relative levels of calreticulin, a number of HTLV-1-infected T
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Fig. 3. Western immunoblot analysis of calreticulin expression levels in selected cell types. Equal protein amounts of whole cell lysates from T cells (Jurkat), astrocytes (U-87 MG), epithelial cells (293T and HeLa), and monocytes (U-937) were subjected to electrophoresis on a 10% TriseHCl SDS polyacrylamide gel and then blotted onto nitrocellulose membrane. Calreticulin expression was detected using a polyclonal antibody.
cell lines (MT-2, C8166, HuT 102, and SLB-1) have been analyzed with respect to calreticulin expression and as compared to calreticulin levels in a non-infected T cell line Jurkat. Interestingly, all the infected cell lines demonstrated higher levels of calreticulin expression compared to the low levels detected in Jurkat cells (Fig. 4). These results suggest that during HTLV-1-mediated transformation of T cells, calreticulin expression would increase facilitating export of Tax from the nucleus, leading to accumulation in the cytoplasm. This process could lead to the stimulation of the NF-kB signaling pathway. Consistent with this hypothesis, constitutive expression of NF-kB by Tax is directly related to T cell transforming properties of Tax [4e6,8].
4. Discussion
Fig. 2. Localization of Tax with calreticulin. Tax-GFP and FLAG-CRT plasmids were transfected into BHK-21 cells. Subsequent to transfection, cells were fixed and visualized. A representative image of the entire population of cells is shown. Colors represent Tax-GFP (green), FLAG-CRT (red), and co-localization of both Tax-GFP and FLAG-CRT (blue). Cells were viewed on an inverted microscope at a magnification of 60. The image was deconvolved using the nearest neighbors method within Slidebook (Intelligent Imaging Innovations).
Herein, we have identified calreticulin as a partner for nuclear export of Tax. Previous studies have reported that proteins containing leucine-rich NESs, such as Tax, are classically known to interact with the nuclear export protein CRM-1 [24,26]. However, consistent with the observations of other exported proteins such as PKI and glucocorticoid receptor (GR), Tax is not exported from the nucleus exclusively through the CRM-1 pathway, demonstrated by the insensitivity to the CRM-1 inhibitor LMB [22,34]. It was logical to infer the presence of an additional receptor for nuclear transport of proteins with hydrophobic export signals. Recently, the calcium binding protein calreticulin was demonstrated to function as a nuclear export receptor, functioning similarly to CRM-1 by transporting substrates through interactions that involve bulky hydrophobic residues within the NES [25,27].
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Fig. 4. Western immunoblot analysis of calreticulin expression levels in noninfected and HTLV-1-infected T cell lines. Equal protein amounts of whole cell lysates were subjected to electrophoresis on a 10% TriseHCl SDS polyacrylamide gel and then blotted onto nitrocellulose membrane. Calreticulin expression was detected using a polyclonal antibody.
This observation is consistent with previous results reported with respect to the characterization of the Tax NES [22]. An important finding that has emerged from these investigations is that Tax has been shown to interact with calreticulin. We have purified calreticulin through immunoprecipitation based on its ability to interact with Tax. Additional evidence demonstrated that Tax and calreticulin co-localized at the nuclear membrane. These observations suggest that calreticulin may serve as a possible binding partner and a control checkpoint in the process of Tax nuclear export. The possibility that calreticulin controls the nuclear export of Tax adds an additional level of complexity. Previous studies have shown that calreticulin can respond dramatically to the Ca2þ activation level of the cell [31]. Calreticulin itself contains several areas throughout its tertiary structure where Ca2þ can bind directly and alter the function of the protein. This is particularly interesting with respect to the nuclear export capabilities of calreticulin. For example, when the low-affinity Ca2þ binding sites are saturated with Ca2þ, calreticulin does not support nuclear export of leucine-rich NEScontaining proteins [31]. However, when Ca2þ was removed by treatment of EGTA, nuclear export of a leucine-rich NES-containing protein was restored. Whether this has a direct effect on the nuclear export and nucleocytoplasmic ratio of Tax has yet to be determined. Studies are needed to further characterize the interaction between calreticulin and Tax and to definitively determine if and under what conditions Tax nuclear export is controlled by calreticulin. When defining the functional roles Tax may have in the cytoplasm and whether regulation of intracellular Tax distribution impacts the pathogenesis of HTLV-1, it is important to note that the nucleocytoplasmic distribution of Tax may be
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dramatically different when compared between different cell types. Furthermore, calreticulin could also define a more specialized protein export pathway that is responsive to the physiological state of the cell. For example, previous studies have shown that calreticulin expression increases in response to heat shock [35], possibly increasing the amount of cytoplasmic calreticulin, which could mediate nuclear export of proteins that are important for the cellular stress response. In addition, both Tax-transfected and HTLV-1-infected astrocytes have been shown to contain a substantial level of Tax within the cytoplasm [33]. Cell type differences in the intracellular localization of Tax may account for differential efficiencies of Tax transactivation, viral replication, and viral pathogenesis within specific cell populations. Altering Tax shuttling between the nucleus and the cytoplasm could alter the balance of ATF/CREB versus NF-kB-mediated responses in the cell. The results of this study have suggested that calreticulin may play a role in shuttling Tax into the cytoplasm, thus activating NF-kB-mediated gene expression and inducing T cell transformation, leading to ATL progression. In addition, the findings also have implications in HAM/TSP pathogenesis. We demonstrated that calreticulin was abundantly expressed in U-87 MG astrocytic cells, a cell type that may be involved in Tax secretion in vivo. HTLV-1-infected cells were also demonstrated to contain an elevated level of calreticulin expression suggesting that this protein may play a role in HTLV-1 pathogenesis. Acknowledgments These studies were supported by United States Public Health Service/National Institutes of Health Grant CA54559 awarded to B. Wigdahl and by funding provided by the Pennsylvania State University College of Medicine Tobacco Settlement Block Grant under the Department of Health’s Health Research Formula Funding Program (State of PA, Act 200177-part of the PA Tobacco Settlement Legislation). References [1] Grassmann R, Berchtold S, Radant I, Alt M, Fleckenstein B, Sodroski JG, et al. Role of human T-cell leukemia virus type 1 region proteins in immortalization of primary human lymphocytes in culture. J Virol 1992;66:4570e5. [2] Sodroski J, Rosen C, Goh WC, Haseltine W. A transcriptional activator protein encoded by the x-lor region of the human T-cell leukemia virus. Science 1985;228:1430e4. [3] Sodroski JG, Rosen CA, Haseltine WA. Trans-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells. Science 1984;225:381e5. [4] Harhaj EW, Good L, Xiao G, Sun SC. Gene expression profiles in HTLVI-immortalized T cells: deregulated expression of genes involved in apoptosis regulation. Oncogene 1999;18:1341e9. [5] Harhaj EW, Sun SC. IKKgamma serves as a docking subunit of the IkappaB kinase (IKK) and mediates interaction of IKK with the human T-cell leukemia virus Tax protein. J Biol Chem 1999;274:22911e4. [6] Jin DY, Giordano V, Kibler KV, Nakano H, Jeang KT. Role of adapter function in oncoprotein-mediated activation of NF-kappaB. Human T-cell leukemia virus type I Tax interacts directly with IkappaB kinase gamma. J Biol Chem 1999;274:17402e5.
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Original article
Interaction of HTLV-1 Tax protein with calreticulin: Implications for Tax nuclear export and secretion Timothy Alefantis a, Katherine E. Flaig b, Brian Wigdahl b, Pooja Jain b,* b
a Vital Probes, Inc., 1300 Old Plank Road, Mayfield, PA 18433, USA Department of Microbiology and Immunology, and Center for Molecular Virology and Neuroimmunology, Center for Cancer Biology, Institute of Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA 19102, USA
Received 25 January 2007; accepted 6 February 2007 Available online 9 March 2007
Abstract Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP). The HTLV-1 transcriptional transactivator protein Tax plays an integral role in virus replication and disease progression. Traditionally, Tax is described as a nuclear protein where it performs its primary role as a transcriptional transactivator. However, recent studies have clearly shown that Tax can also be localized to the cytoplasm where it has been shown to interact with a number of host transcription factors most notably NF-kB, constitutive expression of which is directly related to the T cell transforming properties of Tax in ATL patients. The presence of a functional nuclear export signal (NES) within Tax and the secretion of full-length Tax have also been demonstrated previously. Additionally, release of Tax from HTLV-1-infected cells and the presence of cell-free Tax was demonstrated in the CSF of HAM/TSP patients suggesting that the progression to HAM/TSP might be mediated by the ability of Tax to function as an extracellular cytokine. Therefore, in both ATL and HAM/TSP Tax nuclear export and nucleocytoplasmic shuttling may play a critical role, the mechanism of which remains unknown. In this study, we have demonstrated that the calcium binding protein calreticulin interacts with Tax by co-immunoprecipitation. This interaction was found to localize to a region at or near the nuclear membrane. In addition, differential expression of calreticulin was demonstrated in various cell types that correlated with their ability to retain cytoplasmic Tax, particularly in astrocytes. Finally, a comparison of a number of HTLV-1-infected T cell lines to non-infected T cells revealed higher expression of calreticulin in infected cells implicating a direct role for this protein in HTLV-1 infection. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: HTLV-1 Tax; Nucleocytoplasmic shuttling; Calreticulin
1. Introduction HTLV-1 is the etiologic agent of two major diseases; a progressive lymphoma designated adult T cell leukemia (ATL) and a debilitating neurological disease known as
* Corresponding author. Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, 18 th floor, New College Building, Room #18311, Drexel University College of Medicine, 245 North 15 th Street, Philadelphia, PA 19102, USA. Tel.: þ1 215 762 8586; fax: þ1 215 762 1955. E-mail address: [email protected] (P. Jain). 0753-3322/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2007.02.005
HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 oncoprotein Tax has been studied extensively with respect to its role in regulating cellular and viral gene expression during the course of HTLV-1 infection [1e3]. Specifically, within the context of the viral life cycle, Tax interacts with host transcriptional machinery, especially the ATF/ CREB pathway, to facilitate the binding of selected cellular transcription factors to the viral long terminal repeat (LTR) within the nucleus. In addition to the ATF/CREB pathway, Tax also interacts with the NF-kB signaling pathway in the cytoplasm to induce NF-kB translocation to the nucleus by binding to IKKg, a subunit of the IKK complex [4e7]. The ability of Tax to induce
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the NF-kB pathway has been intensely studied because of its possible role in cellular transformation and the onset of ATL in HTLV-1-infected individuals [4e6,8]. It is unknown whether Tax that interacts with NF-kB in the cytoplasm represents newly synthesized protein resident in the cytoplasm, or whether it represents protein shuttled from the nucleus back to the cytoplasm. If it is the latter, or a combination of the two, the regulation of Tax nucleocytoplasmic shuttling is important since the differential availability of Tax to the NF-kB as compared to ATF/CREB pathway could be expected to have different effects on cellular metabolism and progression of HTLV-1-associated pathogenesis. In addition to its intracellular functions, Tax is also known to cause a variety of effects on cells as an extracellular protein particularly in the context of HAM/TSP. The presence of antibodies against Tax is demonstrated in a majority of HTLV-1-infected individuals [9,10]. Recently, cell-free Tax has been detected in the CSF of HAM/TSP patients [11]. These observations, along with a number of studies including our own, demonstrating the effects of extracellular Tax on both lymphoid and non-lymphoid cells, suggest that Tax is available for immune recognition and can function as an extracellular cytokine [12e 21]. However, it is not yet certain whether the cell-free Tax was the result of apoptosis or necrosis of HTLV-1-infected cells or if it was secreted from the infected cell populations. Consistent with the concept of cellular secretion, the presence of a leucine-rich NES between amino acids 188e200 of Tax has been reported [22]. The secretion of Tax has also been reported from HTLV-1-infected and Tax-transfected cells [16,23]. At any given time, an HTLV-1-infected cell may contain a rate-limiting amount of Tax. Thus, an alteration in the balance of Tax nucleocytoplasmic shuttling may have functional impact on the Tax-induced signaling pathways in both the nucleus and cytoplasm. Furthermore, we hypothesize that Tax may localize to the cytoplasm as an intermediary step during the course of its release from HTLV-1-infected cells, as demonstrated by the presence of Tax in cytoplasmic secretory-like vesicles [23]. Therefore, understanding the mechanism(s) of Tax nuclear export and nucleocytoplasmic shuttling will be critical in clarifying the overall processes associated with HTLV-1-associated immune and central nervous system disease. Traditionally, nuclear export of proteins containing a leucine-rich NES has been shown to be mediated by CRM-1 nuclear export receptor via the nuclear pore complex [24e26]. Previous observations have indicated that the Tax NES (tNES) was shown to function in a CRM-1-dependent manner when fused as an isolated element directly to green fluorescent protein (GFP), the same was not true when the tNES was within the context of full-length Tax [22]. The nucleocytoplasmic localization of full-length Tax-GFP was not altered following treatment of transfected cells with leptomycin B (LMB), an inhibitor of CRM-1 nuclear export. Similarly, studies investigating the nuclear export of a leucine-rich NES containing protein, protein kinase inhibitor (PKI), demonstrated that in a system with CRM-1-depleted cytosol, nuclear export of PKI was retained [27]. These studies have strongly suggested that there are additional export receptors present in mammalian cells that can mediate a CRM-1-independent mechanism
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for nuclear export. Recently the calcium-binding protein implicated in protein folding in the ER lumen, calreticulin, was demonstrated to act as a nuclear export protein [25,27,28]. Calreticulin was demonstrated to facilitate the nuclear export of PKI by a pathway independent of CRM-1 [25]. Several investigators have reported the presence of calreticulin in the nucleus and the cytoplasm [29,30]. Both CRM-1 and calreticulin bind to transport substrates that contain an NES, and require export receptor-substrate-RanGTP complex for export [25]. It has been shown that like HTLV-1 Tax, nuclear export of PKI was not inhibited by LMB. The results presented herein begin to examine the role of calreticulin in Tax nuclear export. Specifically, this investigation demonstrated that calreticulin and Tax interact and co-localize at or near the nuclear membrane in baby hamster kidney (BHK-21) cells, indicating nuclear transport of Tax. Additionally, four HTLV-1-infected T lymphocyte cell lines were demonstrated to contain high levels of calreticulin expression as compared to non-infected Jurkat T lymphocytes. The difference between cells with high levels of cytoplasmic Tax versus those with low levels may be explained by the expression level or relative availability of calreticulin. Therefore, it has been hypothesized that the balance of Tax import and calreticulindependent export from the nucleus provides the cell with a mechanism that may play an important role in regulating Tax biological activity and HTLV-1 induced disease progression. 2. Materials and methods 2.1. Cell culture 293T and BHK-21 cells were grown in Dulbecco‘s modified Eagle’s media. U-87 MG and HeLa cells were grown in Eagle’s minimum essential medium. U-937, Jurkat, and HTLV1-infected T cell lines MT-2, C8166, HuT 102, and SLB-1 were grown in RPMI 1640. Growth media for each cell line was supplemented with 10% fetal bovine serum, 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% CO2 at 90% relative humidity. 2.2. Fusion protein construction and plasmid DNA purification Double-stranded oligonucleotides encoding a six-histidine tag was inserted into the carboxy-terminus of the Tax coding sequence using the QuickChange site-directed mutagenesis kit as described by the manufacturer (Stratagene, La Jolla, CA). The cDNA coding sequence of calreticulin (kindly provided by Dr. Bryce Paschal, University of Virginia School of Medicine) was cloned into the 3xFLAG vector (Sigma, St. Louis, MO) using PCR and calreticulin-specific primers (FLAG-CRT). HTLV-1 Tax constructs for fluorescence microscopy were constructed by cloning full-length Tax cDNA coding sequence into a green fluorescent protein construct
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(pEGFP-N1; Clontech, Palo Alto, CA) yielding a construct encoding fusion proteins in which GFP was fused to the carboxyterminus of Tax as described [22]. Plasmid DNA used for screening and automated sequencing was isolated using the Concert miniprep system as described by the manufacturer (Invitrogen, Carlsbad, CA). The nucleotide sequence of all plasmid constructs was confirmed by automated sequencing (DNA Sequencing Laboratory of the Center for Molecular and Functional Genomics in the Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine) and subsequent bioinformatics analysis using Lasergene software (DNASTAR, Madison, WI). Plasmid DNA used for transient transfections was isolated using the HiSpeed Plasmid Midi kit as described by the manufacturer (Qiagen, Valencia, CA). 2.3. Transient transfections For immunoprecipitation studies, 293T cells were plated in 6-well plates at 1 106 cells per well 24 h prior to transfection. Transfections were performed using 2.4 mg of DNA and Fugene6 transfection reagent (Roche Diagnostics, Mannheim, Germany) with an optimized protocol as described by the manufacturer. For microscopic analyses, BHK-21 cells were plated in an 8-well chamber slide coated with collagen (BD Labware, Bedford, MA) at a concentration of 5 104 cells per well 24 h prior to transfection. Transfections were performed using Fugene6 transfection reagent as described by the manufacturer. 2.4. Immunoprecipitation After transfection (24 h), media was removed and cells were washed once with cold PBS (1 ml). Cells were then lysed and the cell lysate was harvested using the Sigma FLAG immunoprecipitation kit (Sigma) as described by the manufacturer. Anti-FLAG agarose beads were then added to each lysate and the immunoprecipitation was performed as described by the manufacturer (Sigma). After immunoprecipitation (2 h), anti-FLAG agarose beads were washed three times with wash buffer (Sigma), the protein of interest was eluted, and analyzed by western immunoblot analyses (described below) using anti-His6 antibody (ab-9180, 1:6000 dilution, Abcam Ltd, UK).
were then added along with Fluormount-G mounting media (Southern Biotechnology Associates, Inc, Birmingham, AL). Cells were visualized using an Olympus IX-81 automated microscope equipped with the appropriate filter cubes for visualizing Cy-3, and 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-1Ghz processor computer (Apple, Cupertino, CA). Raw fluorescent images were deconvolved (nearest-neighbors method) using Slidebook. 2.6. Whole cell extract preparation Whole cell extracts from Jurkat, 293T, U-87 MG, HeLa, U937 and a number of HTLV-1-infected T cell lines (MT-2, C8166, HuT 102, and SLB-1) were prepared using RIPA buffer [NaCl (150 mM), Igepal (1.0%), deoxycholate (0.5%), Tris pH 8.0 (50 mM)]. Cell debris was cleared from the lysate by centrifugation (5 min, 16,000 g). The protein concentration for each whole cell extract was determined using the DC protein assay as described by the manufacturer (Bio-Rad Laboratories, Hercules, CA). 2.7. Western immunoblot analyses Cell lysates from various cell lines were resuspended in SDS loading buffer and heated at 95 C for 5 min. After heating, samples were loaded onto a 10% TriseHCl SDS polyacrylamide gel (Bio-Rad) and subjected to electrophoresis (180 V, 45 min). Samples were then blotted onto nitrocellulose membrane (Pall Corporation, Ann Arbor, MI) for 2 h (40 mA). Blots were blocked with 5% milk dissolved in a solution of phosphate-buffered saline with 1.0% Tween-20 (PBST, 15 min) and then rinsed with PBST (once for 10 min and twice for 5 min). Blots were incubated with anti-calreticulin antibody (NB 600-101B1, 1:2000 dilution, Novus Biologicals, Littleton, CO), for 1 h at room temperature, rinsed with PBST (once for 10 min and twice for 5 min), and incubated (1 h) with an anti-rabbit horseradish peroxidase-conjugated (HRP) secondary antibody (1:2000 dilution, Amersham Pharmacia, UK). Finally, blots were washed again with PBST (once for 10 min and twice for 5 min) and developed using the Western Lightning kit (Perkin-Elmer Life Sciences, Boston, MA) and exposed to X-ray film.
2.5. Microscopic analyses 3. Results Transfected cells were prepared for microscopy approximately 24 h post-transfection. Cells were fixed for 30 min with paraformaldehyde (4.0%) and washed twice with PBS for 5 min. Cells were then permeabilized with Triton-X 100 (0.5%) in PBS for 15 min and again washed twice for 5 min with PBS. Cells were blocked for 30 min with PBS-BSA (3.0%). FLAG epitope constructs were immunostained using an anti-FLAG-Cy3 antibody (1:1000 dilution in BSA-PBS, 1 h, Sigma, St. Louis, MO). After incubation with antibody, cells were washed three times for 5 min with PBS. Cover slips
3.1. Tax and calreticulin interaction detected by immunoprecipitation Since calreticulin was previously determined to facilitate nuclear export of a leucine-rich NES-containing protein, it was of interest to determine if it could also facilitate the nuclear export of Tax [22,25,31]. Therefore, we have analyzed the interaction of calreticulin and Tax within the context of live cells. An immunoprecipitation assay using 293T cells
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transfected with plasmid constructs encoding either Tax-His6 or FLAG-CRT or a combination of both was utilized. After transfection (24 h), the cell lystes were harvested and antiFLAG agarose beads were utilized to immunoprecipitate FLAG-CRT. Co-immunoprecipitation of Tax-His6 was then detected by western immunoblot analyses using an anti-His6 antibody. As shown in Fig. 1, a band of the predicted 42 kD size of Tax was evident where cells overexpressed both Tax and calreticulin. The negative control samples containing cells transfected with only Tax-His6 or FLAG-CRT did not show a His6-positive band, indicating that the observed immunoprecipitated band was the result of Tax and calrecticulin interaction in intact cells (Fig. 1).
3.2. Tax co-localizes with calreticulin to the nuclear membrane or regions of close proximity Levels of intracellular calreticulin localization differ between cellular compartments due to its multiple functions. While most calrecticulin has been shown to localize to the endoplasmic reticulum based on its role as a molecular chaperone, it has also been shown to accumulate in areas such as the nucleus and secretory pathway. To detail the interaction of Tax with calrecticulin, it was necessary to examine the localization of Tax and calrecticulin within cells. To this end,
Fig. 1. Co-immunoprecipitation of Tax and calreticulin. Cell lysates of TaxHis6-transfected 293T cells were subjected to immunoprecipitation using anti-FLAG agarose beads. Subsequently, co-immunoprecipitated proteins were subjected to electrophoresis on a 10% TriseHCl SDS polyacrylamide gel and then blotted onto nitrocellulose membrane. His6-tag-containing proteins were detected using a His6 polyclonal antibody. The band located at 42 kD represents Tax-His6 co-immunoprecipitated with FLAG-CRT.
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a model cell line BHK-21 (ideal for imaging and localization studies) was co-transfected with Tax-GFP and FLAG-CRT. After 24 h, the cells were fixed and stained with an antiFLAG-Cy3 antibody. A representative image of a Tax-GFP and FLAG-CRT co-transfected BHK-21 that was deconvolved utilizing the nearest neighbors method is shown in Fig. 2. In this image, both Tax-GFP and FLAG-CRT are localized throughout the cell, but are concentrated in an area around the nuclear membrane. Co-localization of these proteins was determined utilizing overlapping masks constructed for both Tax-GFP and FLAG-CRT. Tax-GFP and FLAG-CRT co-localized to an area around the nucleus consistent with the localization at the nuclear membrane and possibly to the nuclear pores, a position that may facilitate Tax egress from the nucleus. These results provide evidence suggesting that Tax interacts with calrecticulin at the nuclear membrane during nucleocytoplasmic transport. 3.3. Endogenous expression of calreticulin differs among various cell types The nucleocytoplasmic distribution of Tax in Jurkat and other T cell lines has been demonstrated in a recent report by Azran et al. [32]. However, cellular distribution of Tax may be different when compared between different cell types, or within specific cell populations at various stages of the cell cycle. For example, both Tax-transfected and HTLV-1-infected astrocytes have been shown to contain a substantial amount of Tax within the cytoplasm [33]. The amount of cytoplasmic Tax can also differ significantly based on the method of Tax expression. For example, Tax was detected specifically in the nucleus of JPX cells (a Jurkat-derived T cell line that stably expresses Tax) and primarily in the cytoplasm of T cells derived from PBMCs of an ATL patient [33]. Cell type differences such as these may account for differential efficiencies of Tax transactivation, viral replication, and overall HTLV-1 pathogenesis. We hypothesized that if calrecticulin is a critical mediator of Tax nuclear export, its endogenous expression in various cell types could account for observed differences in cytoplasmic Tax in various cell types. Therefore, calreticulin expression was examined in various cell types including T cell line (Jurkat), astrocytes (U-87 MG), epithelial cells (293T and HeLa), and monocytes (U-937) by western immunoblot analysis (Fig. 3). It was interesting to note that epithelial cells 293T and HeLa, and astrocytic cell line (U-87 MG) demonstrated higher expression levels of calreticulin as compared to T cells and monocytes for the same amount of total protein used in the analysis. In agreement with previous studies, these results suggest a correlation between higher levels of calreticulin in cell types shown to contain higher levels of cytoplasmic Tax such as astrocytes [33]. 3.4. Higher expression of calreticulin in HTLV-1-infected T cells compared to non-infected cells To correlate these observations with HTLV-1 infection and relative levels of calreticulin, a number of HTLV-1-infected T
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Fig. 3. Western immunoblot analysis of calreticulin expression levels in selected cell types. Equal protein amounts of whole cell lysates from T cells (Jurkat), astrocytes (U-87 MG), epithelial cells (293T and HeLa), and monocytes (U-937) were subjected to electrophoresis on a 10% TriseHCl SDS polyacrylamide gel and then blotted onto nitrocellulose membrane. Calreticulin expression was detected using a polyclonal antibody.
cell lines (MT-2, C8166, HuT 102, and SLB-1) have been analyzed with respect to calreticulin expression and as compared to calreticulin levels in a non-infected T cell line Jurkat. Interestingly, all the infected cell lines demonstrated higher levels of calreticulin expression compared to the low levels detected in Jurkat cells (Fig. 4). These results suggest that during HTLV-1-mediated transformation of T cells, calreticulin expression would increase facilitating export of Tax from the nucleus, leading to accumulation in the cytoplasm. This process could lead to the stimulation of the NF-kB signaling pathway. Consistent with this hypothesis, constitutive expression of NF-kB by Tax is directly related to T cell transforming properties of Tax [4e6,8].
4. Discussion
Fig. 2. Localization of Tax with calreticulin. Tax-GFP and FLAG-CRT plasmids were transfected into BHK-21 cells. Subsequent to transfection, cells were fixed and visualized. A representative image of the entire population of cells is shown. Colors represent Tax-GFP (green), FLAG-CRT (red), and co-localization of both Tax-GFP and FLAG-CRT (blue). Cells were viewed on an inverted microscope at a magnification of 60. The image was deconvolved using the nearest neighbors method within Slidebook (Intelligent Imaging Innovations).
Herein, we have identified calreticulin as a partner for nuclear export of Tax. Previous studies have reported that proteins containing leucine-rich NESs, such as Tax, are classically known to interact with the nuclear export protein CRM-1 [24,26]. However, consistent with the observations of other exported proteins such as PKI and glucocorticoid receptor (GR), Tax is not exported from the nucleus exclusively through the CRM-1 pathway, demonstrated by the insensitivity to the CRM-1 inhibitor LMB [22,34]. It was logical to infer the presence of an additional receptor for nuclear transport of proteins with hydrophobic export signals. Recently, the calcium binding protein calreticulin was demonstrated to function as a nuclear export receptor, functioning similarly to CRM-1 by transporting substrates through interactions that involve bulky hydrophobic residues within the NES [25,27].
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Fig. 4. Western immunoblot analysis of calreticulin expression levels in noninfected and HTLV-1-infected T cell lines. Equal protein amounts of whole cell lysates were subjected to electrophoresis on a 10% TriseHCl SDS polyacrylamide gel and then blotted onto nitrocellulose membrane. Calreticulin expression was detected using a polyclonal antibody.
This observation is consistent with previous results reported with respect to the characterization of the Tax NES [22]. An important finding that has emerged from these investigations is that Tax has been shown to interact with calreticulin. We have purified calreticulin through immunoprecipitation based on its ability to interact with Tax. Additional evidence demonstrated that Tax and calreticulin co-localized at the nuclear membrane. These observations suggest that calreticulin may serve as a possible binding partner and a control checkpoint in the process of Tax nuclear export. The possibility that calreticulin controls the nuclear export of Tax adds an additional level of complexity. Previous studies have shown that calreticulin can respond dramatically to the Ca2þ activation level of the cell [31]. Calreticulin itself contains several areas throughout its tertiary structure where Ca2þ can bind directly and alter the function of the protein. This is particularly interesting with respect to the nuclear export capabilities of calreticulin. For example, when the low-affinity Ca2þ binding sites are saturated with Ca2þ, calreticulin does not support nuclear export of leucine-rich NEScontaining proteins [31]. However, when Ca2þ was removed by treatment of EGTA, nuclear export of a leucine-rich NES-containing protein was restored. Whether this has a direct effect on the nuclear export and nucleocytoplasmic ratio of Tax has yet to be determined. Studies are needed to further characterize the interaction between calreticulin and Tax and to definitively determine if and under what conditions Tax nuclear export is controlled by calreticulin. When defining the functional roles Tax may have in the cytoplasm and whether regulation of intracellular Tax distribution impacts the pathogenesis of HTLV-1, it is important to note that the nucleocytoplasmic distribution of Tax may be
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dramatically different when compared between different cell types. Furthermore, calreticulin could also define a more specialized protein export pathway that is responsive to the physiological state of the cell. For example, previous studies have shown that calreticulin expression increases in response to heat shock [35], possibly increasing the amount of cytoplasmic calreticulin, which could mediate nuclear export of proteins that are important for the cellular stress response. In addition, both Tax-transfected and HTLV-1-infected astrocytes have been shown to contain a substantial level of Tax within the cytoplasm [33]. Cell type differences in the intracellular localization of Tax may account for differential efficiencies of Tax transactivation, viral replication, and viral pathogenesis within specific cell populations. Altering Tax shuttling between the nucleus and the cytoplasm could alter the balance of ATF/CREB versus NF-kB-mediated responses in the cell. The results of this study have suggested that calreticulin may play a role in shuttling Tax into the cytoplasm, thus activating NF-kB-mediated gene expression and inducing T cell transformation, leading to ATL progression. In addition, the findings also have implications in HAM/TSP pathogenesis. We demonstrated that calreticulin was abundantly expressed in U-87 MG astrocytic cells, a cell type that may be involved in Tax secretion in vivo. HTLV-1-infected cells were also demonstrated to contain an elevated level of calreticulin expression suggesting that this protein may play a role in HTLV-1 pathogenesis. Acknowledgments These studies were supported by United States Public Health Service/National Institutes of Health Grant CA54559 awarded to B. Wigdahl and by funding provided by the Pennsylvania State University College of Medicine Tobacco Settlement Block Grant under the Department of Health’s Health Research Formula Funding Program (State of PA, Act 200177-part of the PA Tobacco Settlement Legislation). References [1] Grassmann R, Berchtold S, Radant I, Alt M, Fleckenstein B, Sodroski JG, et al. Role of human T-cell leukemia virus type 1 region proteins in immortalization of primary human lymphocytes in culture. J Virol 1992;66:4570e5. [2] Sodroski J, Rosen C, Goh WC, Haseltine W. A transcriptional activator protein encoded by the x-lor region of the human T-cell leukemia virus. Science 1985;228:1430e4. [3] Sodroski JG, Rosen CA, Haseltine WA. Trans-acting transcriptional activation of the long terminal repeat of human T lymphotropic viruses in infected cells. Science 1984;225:381e5. [4] Harhaj EW, Good L, Xiao G, Sun SC. Gene expression profiles in HTLVI-immortalized T cells: deregulated expression of genes involved in apoptosis regulation. Oncogene 1999;18:1341e9. [5] Harhaj EW, Sun SC. IKKgamma serves as a docking subunit of the IkappaB kinase (IKK) and mediates interaction of IKK with the human T-cell leukemia virus Tax protein. J Biol Chem 1999;274:22911e4. [6] Jin DY, Giordano V, Kibler KV, Nakano H, Jeang KT. Role of adapter function in oncoprotein-mediated activation of NF-kappaB. Human T-cell leukemia virus type I Tax interacts directly with IkappaB kinase gamma. J Biol Chem 1999;274:17402e5.
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