T-Cell Growth Transformation by Herpesvirus Saimir
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病菌学杂志 2005年第9期
Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universitt Erlangen-Nürnberg, D-91054 Erlangen, Germany
ABSTRACT
Herpesvirus saimiri (saimirine herpesvirus 2) (HVS), a T-lymphotropic tumor virus, induces lymphoproliferative disease in several species of New World primates. In addition, strains of HVS subgroup C are able to transform T cells of Old World primates, including humans, to permanently growing T-cell lines. In concert with the Stp oncoprotein, the tyrosine kinase-interacting protein (Tip) of HVS C488 is required for T-cell transformation in vitro and lymphoma induction in vivo. Tip was previously shown to interact with the protein tyrosine kinase Lck. Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis and has also been detected in HVS-transformed T-cell lines. Furthermore, Tip contains a putative consensus YXPQ binding motif for the SH2 (src homology 2) domains of STAT1 and STAT3. Tip tyrosine phosphorylation at this site was required for binding of STATs and induction of STAT-dependent transcription. Here we sought to address the relevance of STAT activation for transformation of human T cells by introducing a tyrosine-to-phenylalanine mutation in the YXPQ motif of Tip of HVS C488. Unexpectedly, the recombinant virus was still able to transform human T lymphocytes, but it had lost its capability to activate STAT3 as well as STAT1. This demonstrates that growth transformation by HVS is independent of STAT3 activation.
INTRODUCTION
Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that were originally discovered as key mediators of cytokine signaling. They are involved in signaling by the interleukin-6 (IL-6) family cytokines, the IL-2 family cytokines, and numerous growth factors, including epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor 1. Latent inactive STATs reside in the cytoplasm; in response to receptor activation, they are recruited to a receptor where they are tyrosine phosphorylated by receptor tyrosine kinases (RTK) or receptor-associated tyrosine kinases, such as JAK or Trk kinases (reviewed in references 36, 42, 46, and 48). Active STAT dimers are then formed via the reciprocal interaction between the SH2 domain of one monomer and the phosphorylated tyrosine of the other. Activated dimers translocate to the nucleus, where they bind to specific DNA response elements in the promoters of target genes and activate transcription. Furthermore, various oncoproteins can activate specific STAT molecules (especially STAT3 and STAT5) (8, 15), and inappropriate STAT activation directly contributes to oncogenesis by stimulating cell proliferation and preventing apoptosis. Constitutive activation of several STATs was frequently detected in a wide range of human cell lines and primary tumors, including lymphoid malignancies (lymphomas, leukemias, and multiple myelomas) (30, 63, 67). Constitutively activated mutant STAT3 and STAT5 proteins have been shown to possess transforming properties and to be strongly associated with tumor development and progression (7, 9).
Herpesvirus saimiri (HVS), the prototype of the rhadinoviruses (gamma-2-herpesviruses), regularly establishes lifelong persistent infections in squirrel monkeys (Saimiri sciureus) of South American rainforests. While it does not cause disease in its natural host (54), experimental infection with HVS causes acute peripheral T-cell lymphoma in other New World primate species, such as tamarins, common marmosets, or owl monkeys (29). HVS strains were classified into three subgroups (A, B, and C) according to pathogenic properties and sequence variation in the left-terminal nonrepetitive genomic region (18, 52). Certain subgroup C strains are able to stimulate human T lymphocytes to stable antigen-independent growth in culture (4).
The genetic determinants responsible for induction of T-cell leukemia and T-cell transformation in vitro are located in the variable region at the left end of the HVS low GC content DNA (13, 17, 19, 21, 45, 55). At this position, virus strains of subgroup C have two open reading frames, termed stpC (saimiri transformation-associated protein of subgroup C strains) and tip (tyrosine kinase-interacting protein), which are transcribed into a single bicistronic mRNA (5, 6, 25, 38). While not required for viral replication, deletion of either stpC or tip abolishes transformation by HVS in vitro and pathogenicity in vivo (21, 22, 44, 51). StpC was shown to interact with the small G-protein Ras and stimulated mitogen-activated protein kinase activity (39). StpC further interacts with tumor necrosis factor receptor-associated factors, leading to nuclear factor kappa B (NF-B) activation (47).
Tip was identified as a 40-kDa phosphoprotein that coprecipitated with the T-cell-specific nonreceptor tyrosine kinase p56lck from lysates of C488-transformed T cells (6). The Tip kinase-interacting domain consists of two motifs, nine amino acids with homology to the C-terminal regulatory regions of various Src kinases (CSKH) and a proline-rich SH3 domain-binding sequence (SH3B), both of which are required for the interaction with the kinase (2, 6, 40). Tip is a substrate for the tyrosine kinase Lck (6, 40) and contains several tyrosine residues, three of which are conserved in all virus strains investigated (24, 31). Tip binding to Lck was reported to activate the enzyme (26, 34, 49, 64). On the other hand, in fibroblasts transformed with a constitutively active mutant of Lck, overexpression of Tip of strain C488 reversed the transformed phenotype. This was taken as evidence that Tip can downmodulate Lck-dependent signaling. These effects were even more pronounced when Tip Y114 was mutated to serine (Y114S) (32, 41).
In addition to Lck, coprecipitation of phosphorylated STAT1 and STAT3 with Lck and Tip-C484 was reported (50). A YXPQ motif of Tip (Y72; equivalent to Y114 of C488) conforms to a putative consensus binding site for STAT factors (62). Tyrosine 72 phosphorylation was required for STAT binding and transcription activation (33). This has also been demonstrated for the equivalent Y114 of strain C488 (43).
The published data were generated by overexpressing Tip in epithelial cells or T-cell lines, but the role of STAT activation by Tip in T-cell growth transformation has remained unclear. In the course of identifying the Tip elements required for transformation of human T cells, we addressed the question of whether constitutive activation of STAT3 plays a decisive role. A mutation of tyrosine 114 was introduced into recombinant viruses. This allowed us to study the behavior of Tip in the viral background, specifically to investigate the transforming potential on human cord blood lymphocytes (CBL). Surprisingly, a mutation of tyrosine 114 to phenylalanine (Y114F) did not abrogate the transforming ability of HVS and resulted in cell lines devoid of detectable levels of phosphorylated STAT3.
MATERIALS AND METHODS
Generation of recombinant viruses. The HVS strain C488 genome (18, 24) has been cloned into a set of overlapping cosmid clones; recombinant viruses can be reconstituted by cotransfection of overlapping cosmids into permissive owl monkey kidney (OMK) cells (23). To introduce the Y114F mutation into the viral genome, a 5' fragment of tip was PCR amplified from the left terminal cosmid 331 with primers 488stuY114F (5'-AAAGGTACCTCGAGCCTAGGCCTGAATGTGCTAGTTTCATTG-3'), which contained KpnI, XhoI, and StuI sites and 170326 (5'-GTAGTAAACTAAGAGCAAAGCAAGC-3'). The resulting fragment was digested with KpnI-SpeI and ligated into KpnI-SpeI-digested plasmid pSTBlueStpCHN (HVS C488 genome nucleotides 1463 to 2419) to obtain plasmid pSTBlueStpCY114FHN, which contains stpC and the first 338 bp of tip. A XhoI-StuI fragment of a Tip expression plasmid (40) was inserted into plasmid pSTBlueStpCY114FHN, resulting in plasmid pSTBlueStpCTipY114F. Correct insertion was verified by restriction mapping and DNA sequencing. A 1.7-kb Bst1107I fragment (HVS C488 nucleotides 721 to 2416) of pSTBlueStpCTipY114F was reinserted into Bst1107I-digested cosmid 331Bst1107I, resulting in cosmid 331-Y114F. Correct insertion into the cosmid was verified by restriction enzyme mapping. Recombinant viruses were generated by liposome-mediated cotransfection (Lipofectamine; Invitrogen) of a set of overlapping cosmids (23), including the altered cosmid 331-Y114F, into permissive OMK cells. The cosmids were linearized by restriction with NotI before transfection, which also removed the pWE15 cloning vector. The identity and purity of each recombinant virus were confirmed by PCR analysis. The correct tip sequence was verified by DNA sequencing of the complete tip gene with primers 110857/Tip-c3 (5'-CTCAGGCATCTTTCTTTGCATTTC-3') and 110858/Tip-c5 (5'-GGTGAATCACAAAACAGCACAAAC-3').
Cell culture and lymphocyte transformation. OMK cells (ATCC CRL1556) were used for propagation of HVS. The cells were cultivated in Dulbecco's modified Eagle's medium supplemented with glutamine (350 μg/ml), gentamicin (100 μg/ml), and 10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany). Virus stocks were generated by infection of confluent OMK cells in tissue culture flasks at a low multiplicity of infection. When lysis was complete, cell-free supernatants were stored at –80°C.
Lymphocyte cultures were grown in RPMI 1640 (Invitrogen, Karlsruhe, Germany) and Panserin 401 medium mixed at a ratio of 1:1 supplemented with 10% irradiated fetal calf serum (Pan Biotech, Aidenbach, Germany), glutamine (350 μg/ml), and gentamicin (100 μg/ml).
Human CBL were isolated by selective sedimentation of erythrocytes for 45 min at 37°C in 5% dextran (molecular weight of 250,000) in 150 mM NaCl. These primary cells were stimulated with 1 μg/ml phytohemagglutinin, and exogenous IL-2 (Roche Diagnostics, Mannheim, Germany) (10 units/ml) was added to the cells after 24 h. On the next day, the cells were infected as described previously (27). Five days after infection, cells were split into two cultures, and exogenous IL-2 was depleted from one of the cultures by centrifuging and washing the cells. Cell culture densities were determined by automated cell counting (Micro Cell Counter F-300 [Sysmex, Norderstedt, Germany] and Beckman-Coulter Z2 [Beckman-Coulter, Krefeld, Germany]). Transformation of the resulting T-cell lines was assessed microscopically and by observation of accelerated growth compared with uninfected control cultures, which ceased growing after 4 to 6 weeks. The transformed T-cell lines were analyzed by PCR and sequencing in order to confirm the specific viral genotype in the cells. None of the uninfected controls yielded a transformed T-cell line.
RNA isolation, cDNA synthesis, and amplification. Total cellular RNA was isolated by acidic phenol guanidinium thiocyanate-phenol-chloroform extraction (12). RNA (1.5 μg) was digested in a volume of 12 μl with 10 U of RNase-free DNase I (Roche Diagnostics) in the presence of 1 U of RNaseOUT RNase inhibitor (Invitrogen) and 1 mM dithiothreitol at 37°C for 30 min, followed by a heat inactivation step of 10 min at 70°C. The samples were then divided into two parallel reaction mixtures and processed with the ThermoScript reverse transcription PCR (RT-PCR) system (Invitrogen) in 20-μl reaction mixtures with or without reverse transcriptase according to the supplier's protocol. RNA complementary to the cDNA was removed by addition of 1 U of RNase H (MBI Fermentas) and incubation for 20 min at 37°C. The reaction mixtures were stored at –20°C. A 2-μl sample of the reaction mixtures was used for RT-PCR analysis. PCR conditions were as follows: a 2-min initial denaturation step at 96°C; 39 cycles, with 1 cycle consisting of 10 s at 96°C, 20 s at 62°C, and 40 s at 70°C; a final extension step of 2 min at 70°C; and a 12°C hold. Primers were specific for HVS C488 Tip (TipL, 5'-ATCCCATGTTGCTGACAAGTCACG-3', and TipR, 5'-CAAACACGTCAAGCAGTAGTGGCAG-3') and for -actin (hActin5, 5'-CCAAGGCCAACCGCGAGAAGATGAC-3', and hActin3, 5'-AGGGTACATGGTGGTGCCGCCAGAC-3').
DNA sequence analysis. Nucleotide sequences were determined with an ABI PRISM 3100 genetic analyzer (Applied Biosystems) using the Dye-Deoxy Terminator Sequencing kit according to the manufacturer's instructions (Perkin-Elmer). DNA sequence evaluation was performed with the GAP4 software (Staden Package) (16).
Immunoblotting. Cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP-40, sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with 1 mM sodium orthovanadate (Na3VO4), 5 mM NaF, 10 μg/ml of aprotinin, and 10 μg/ml of leupeptin (Sigma-Aldrich, Taufkirchen, Germany). Total protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL). Cell lysates (20 μg per lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, Mass.). Membranes were incubated at room temperature for 1 h or at 4°C overnight in blocking buffer (phosphate-buffered saline, pH 7.4, 0.1% Tween 20, 5% [wt/vol] nonfat dried milk powder) and then incubated with primary antibody diluted in blocking buffer for 1 hour or overnight. Equal loading and electrotransfer were controlled by Coomassie brilliant blue R-250 (CBB) staining of PVDF membranes after detection of enhanced chemiluminescence. Antibodies against STAT3 (F-2) and phosphorylated-STAT3-Y705 (B-7) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a dilution of 1:1,000. Primary antibodies were detected with murine immunoglobulin-specific, horseradish peroxidase-coupled secondary antibodies (Dako, Hamburg, Germany) at a dilution of 1:1,000 and an enhanced chemiluminescence substrate (Amersham, Freiburg, Germany) by a Fuji LAS-1000 chemiluminescence detection system (Raytest, Straubenhardt, Germany).
STAT activation assay. Activation of STAT1 and STAT3 was determined using a TransAM STAT family transcription factor assay (Active Motif, Carlsbad, CA) according to the manufacturer's recommendations. Cell nuclei were prepared from transformed lymphocytes by hypotonic lysis. Nuclear lysates (8 μg of total protein) were incubated in 96-well dishes containing immobilized oligonucleotides containing a STAT consensus DNA-binding site (5'-TTCCCGGAA-3') for 1 h at room temperature. Wells were then washed three times, and 100 μl of monoclonal antibody (1:1,000 dilution) was added to each well for 1 h at room temperature. Wells were washed three times, and then 100 μl of horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution) was added to each well for 1 h at room temperature. Wells were washed four times, and 100 μl of developing solution was added to each well for 10 min at room temperature. Stop solution (100 μl) was added to each well, and the absorbance at 450 nm was determined using an enzyme-linked immunosorbent assay (ELISA) reader set to 450 nm. Specificity of binding was determined by competition with wild-type and mutated STAT oligonucleotides. Nuclear extracts from HepG2 cells (treated with IL-6, 100 ng/ml) were included as positive controls for STAT3 and nuclear extracts from U-937 cells (treated with tetradecanoyl phorbol acetate and gamma interferon) for STAT1.
Flow cytometry. Transformed human T cells were analyzed by flow cytometry with antibodies for T-cell surface epitopes on a FACScalibur flow cytometer (Becton Dickinson) according to standard protocols. The directly labeled monoclonal antibodies (Cy-Chrome, fluorescein isothiocyanate, or phycoerythrin conjugated) were specific against CD3 (Leu-4), CD4 (Leu-3a), CD8 (Leu-2a), CD25 (M-A251), CD28 (L293), HLA-DR (L243), CD80 (L307.4), and CD86 (IT2.2) (all from BD Biosciences Immunocytometry Systems and Pharmingen). Directly labeled isotype-matched control monoclonal antibodies were used (BD Biosciences Immunocytometry Systems and Pharmingen).
RESULTS
Construction of recombinant viruses. In order to identify the relevant motifs of the Tip protein required for transformation of human T lymphocytes by HVS, we set out to construct recombinant viruses carrying defined mutations in the tip coding sequence. First, we addressed the relevance of STAT factors by mutating the Tip residue responsible for STAT activation. A shuttle plasmid containing the stpC and tip genes was generated, and mutations were introduced by standard cloning and PCR-based mutagenesis. After verification by DNA sequencing, the mutated tip was introduced into a cosmid encompassing the left terminal region of HVS strain C488. In more detail, the respective stpC and tip genes were subcloned into plasmid pSTBlue and the tyrosine residue 114 was replaced by phenylalanine (Y114F). The modified coding sequence was then reinserted into the left terminal cosmid 331Bst1107I, which lacks the stpC and tip genes, to generate cosmid 331-Y114F (Fig. 1). Recombinant virus was then generated by homologous recombination of five overlapping cosmids in permissive OMK cells, which allows construction of recombinant viruses without wild-type virus contamination (23). The viruses M11 (wild-type C488) and two independent recombinant C488-Y114F obtained after cotransfection were verified by PCR, and the tip gene was sequenced.
Transformation of human T cells in vitro. Herpesvirus saimiri C488 is able to transform human T cells to antigen-independent growth in vitro (4). In order to test the transforming potential on human CBL, the CBL were infected in parallel with wild-type C488 or cosmid-generated wild-type M11 control viruses and recombinant Tip-mutated viruses. Proliferation of the cells was compared to that of uninfected controls. Cells were regarded as growth-transformed cells when they had proliferated continuously for at least 3 months while uninfected control cells had ceased growing. Transformed T-cell lines were analyzed by PCR and sequencing in order to confirm the specific viral genotype in the cells.
The uninfected control cells stopped growing after 2 to 3 weeks of culture. Surprisingly, the HVS C488-Y114F mutant was still capable of transforming human T cells to antigen-independent growth in the presence or absence of exogenous IL-2. The experiments were performed with two independently generated C488-Y114F mutants; both recombinants yielded identical results. Moreover, the cells whose growth had been transformed with Tip-Y114F recombinant viruses grew to higher cell numbers than the wild-type HVS C488-transformed cells (Fig. 2 and 3). Thus, growth transformation of human T lymphocytes by HVS is independent of the presence of the tyrosine residue at position 114.
Altered morphology and phenotype of transformed cells. The surface phenotypes of the human CBL-derived T-cell lines successfully transformed with wild-type/M11 and C488-Y114F viruses were analyzed by flow cytometry. Herpesvirus saimiri-transformed T lymphocytes show the surface markers characteristic of mature, activated T cells (4, 27, 28, 53). Figure 4 shows the surface marker expression of HVS C488- and HVS C488-Y114F-transformed cells. Wild-type/M11 and C488-Y114F-transformed cells showed no differences in CD3 and HLA-DR expression. Y114F-transformed cells had a high expression level of CD25, regardless of the presence or absence of exogenous IL-2, and they show a higher CD28 expression compared to wild-type virus-transformed cells. With regard to CD28 ligands, all wild-type virus-transformed cells tested were positive for CD80 (B-7.1) only, whereas C488-Y114F-transformed cell lines expressed both CD80 and CD86 (B-7.2) activation markers. While cultures growth transformed by HVS C488 or M11 wild-type virus consisted of CD4+ cells or CD8+ cells, Tip Y114F-transformed CBL had a CD4+ phenotype; a minor fraction of CD4+ CD8+cells was found in some of the cell lines.
The expression level of Tip could theoretically contribute to these differences, although expression of HVS stpC/tip oncogenes has been found to be highly variable in successfully transformed human T cells (25, 26). As Tip expression in transformed cell lines is hardly detectable by Western blotting, we used RT-PCR to analyze the transcription of tip in T-cell lines transformed by wild-type and Y114F viruses. RT-PCR for tip and -actin was performed on total RNA with appropriate controls, and reaction products were separated by agarose gel electrophoresis. A 413-bp fragment specific for tip (Fig. 5) was detected in all infected cultures. RT-PCR for -actin (587-bp fragment) shows that similar amounts of cellular RNA were used for cDNA synthesis (Fig. 5). According to the result of this experiment, tip transcription is variable, but no consistent difference was seen.
Phosphorylation and activation status of STAT3. Interaction of Tip with Lck and STAT has been investigated for HVS strain C484 in vitro and in transfected cells: phosphorylated STAT1 and STAT3 were immunoprecipitated together with Tip-C484 and Lck from lysates of transfected Jurkat cells (49). It has further been shown that Tip-C484 enhances the activity of STAT1 and STAT3 in the presence of Lck. For activation of STAT-dependent transcription, tyrosine 72 of Tip (identical to Y114 of C488), which is part of a putative consensus binding site (YXPQ) for STAT factors, was required (33).
The phosphorylation of STAT3 in HVS C488-Y114F-transformed cell lines was compared to wild-type HVS-transformed T cells by Western blotting. As to be expected from earlier reports, Y705 of STAT3 was found to be tyrosine phosphorylated in wild-type virus-transformed human T cells. No significant phosphorylation at the regulatory Y705 was detected in HVS C488-Y114F-transformed human T cells, while STAT3 was expressed at approximately equal levels in all cell lines tested (Fig. 6A). In immunoblots with two different Y701 phosphorylation-specific antibodies, STAT1 phosphorylation at the regulatory Y701 was detected only weakly or was absent in wild-type-virus-transformed T cells, and Y114F-transformed cells showed no noticeable STAT1 Y701 phosphorylation (data not shown). In addition, binding of STAT1 and STAT3 to specific DNA recognition sites was analyzed by an ELISA-based assay that allows detection and quantification of DNA-binding activity of transcription factors (Fig. 6B). This assay demonstrated that HVS C488-transformed cells show a weak but specific STAT1 activation, whereas Y114F-transformed T-cell lines show no activation of STAT1. The STAT3 assay showed strong activation in HVS C488-transformed cells, while Y114F cell lines exhibited no STAT3-specific DNA binding and thus no activation at all. The specificity of the assay is monitored by addition of a wild-type consensus oligonucleotide which acts as a competitor. In summary, both tyrosine phosphorylation and DNA-binding activity of STAT1 and STAT3, which were regularly found in wild-type virus-transformed T cells, was not observed in human T cells growth transformed by HVS C488-Y114F.
DISCUSSION
HVS causes rapidly progressive T-cell leukemia and lymphoma in several species of New World primates, while it is not pathogenic in its persistently infected natural host, the squirrel monkey (Saimiri sciureus) (29). Strains of the highly oncogenic subgroup C, particularly strain C488, are also capable of transforming primary human T cells to permanent antigen-independent growth (4). Two open reading frames, stpC and tip, are necessary for the transforming and pathogenic properties of HVS C488 (1, 20).
So far, the essential contribution of Tip to T-cell transformation by HVS was mainly attributed to an Lck-mediated STAT3 activation. This hypothesis was based on the constitutive STAT3 activation found in transformed human and monkey T cells as a consequence of transformation by HVS of subgroup C and also of subgroup A (14, 59, 61), as well as in human cell lines cotransfected with Tip- and Lck-encoding plasmids (33, 43, 50). It was supported by numerous studies showing that improper STAT activation contributes to oncogenesis by stimulation of cell proliferation and inhibition of apoptosis. Moreover, a variety of human cell lines and primary tumors exhibits constitutively active STATs (3, 10, 30, 37, 56, 63, 65, 67).
For Tip of HVS strain C484, it had been shown that phosphorylation of tyrosine 72, which is part of a putative consensus binding site for STAT factors (YXPQ), is required for STAT binding and transcriptional activation (33). The YXPQ interaction motif of Tip is well conserved among different isolates of HVS subgroup C (24, 31). Accordingly, Y114 of Tip-C488, like Y72 of Tip-C484, is required for stimulation of STAT-dependent reporter gene transcription in transiently transfected Jurkat T cells, and mutation of the respective residue results in loss of STAT3 activation (33, 43).
To challenge the hypothesis of an essential function for STAT3 activation in human T-cell transformation by HVS strain C488, we inserted a Tip-Y114F mutation into recombinant viruses. Our data show that two independently generated recombinant HVS C488-Y114F viruses were still able to transform human T cells to antigen-independent growth in culture (Fig. 3). At the same time, constitutive STAT3 phosphorylation and activation observed in wild-type HVS C488 or recombinant cosmid-reconstituted wild-type-virus-transformed cells (Fig. 6) was lost in HVS C488-Y114F-transformed cells. Thus, activation of STAT3 seems to play a minor role in growth transformation of human T cells by HVS.
The question arises whether the process of viral T-cell transformation is completely independent of STAT3 or whether other (STAT) factors can compensate for the missing STAT3 activation. However, STAT1 activation could not be detected in HVS C488-Y114F-transformed cells (Fig. 6B). A contribution of STAT heterodimers, which have been reported for STAT1 and STAT2 heterodimers and STAT1 and STAT3 heterodimers (reviewed in references 35, 46, and 60), is also unlikely, since neither activated STAT1 nor STAT3 was detected in nuclear extracts from HVS C488-Y114F-transformed cells (Fig. 6B).
The phenotype of lymphocytes transformed with the HVS Y114F mutant viruses was slightly different from that of cells carrying wild-type HVS. They grew to higher cell numbers and formed larger aggregates (Fig. 2 and 3). Like T cells transformed with the parental HVS C488 (28), they displayed typical surface markers of mature T cells. It has been reported that overexpression of Tip in T-cell lines results in a downmodulation of CD3 and CD4 surface expression (57), which was more pronounced in Tip-Y114S (32, 58). In our hands, HVS-transformed cells expressing the HVS oncogene tip from the viral promoter do not show such a dramatic effect; instead, they showed only a modestly lower surface expression of CD3 in C488-Y114F-transformed cells compared to HVS-C488-transformed cells and normal expression of CD4 (Fig. 4).
Surface expression of CD25 (IL-2 receptor ) and CD28 were augmented, and in addition to CD80, HVS C488-Y114F-transformed cells displayed the alternative CD28 ligand, CD86. A role of STAT3 in the regulation of these surface markers has not been described so far. The differences in the phenotype might be related to targeting of distinct T-cell populations by wild-type and mutant viruses, respectively. Alternatively, the phenotypic variation could be attributed to differentiation into divergent T-cell subpopulations in the course of transformation, which might also be influenced by STAT3 activity.
In summary, this work provides strong evidence that STAT3 (and STAT1) activation, though consistently found in T-cell lines transformed by HVS strains in vitro or in vivo (14, 59, 61), is not a prerequisite for human primary T-cell transformation to antigen-independent growth by HVS subgroup C strains. Therefore, the essential role of Tip in viral T-cell transformation has to be mediated by effectors other than STAT3. The association of Tip and Lck with the endosomal targeting protein p80 was found to responsible for downregulation of TCR and CD4 surface expression and thereby their signal transduction pathways (57). This seems to be STAT3 independent, since it was found to be even more pronounced with Tip-Y114F. Although we do not see a dramatic TCR complex modulation in transformed human T cells, pathways linked to the endocytic process might be relevant for transformation of T cells by HVS subgroup C strains. However, in general, growth regulatory mechanisms related to receptor endocytosis have not been described yet.
Therefore, we would like to suggest another model of Tip's role in T-cell transformation which largely relies on direct effects of Tip on Lck function. While activation of Lck's in vitro activity upon Tip binding has been reported repeatedly (26, 34, 43, 49, 64), several in vivo approaches argue in favor of kinase inhibition by Tip (32, 41). In a recent publication, Tip-mediated inhibition of T-cell receptor signal transduction was shown to occur at the level of ZAP70 phosphorylation and resulted in a failure to induce intracellular calcium mobilization, while phosphorylation of the T-cell receptor -chain was not affected (11). An attractive hypothesis deduced from these data would include an altered substrate specificity of Lck in the presence of Tip. Thereby, Lck activity may be redirected towards a target not yet identified. Tip may again be involved by recruiting this alternative target molecule to the Lck complex. STAT3 would have been an attractive candidate, but it is excluded by this study. Numerous other proteins not yet characterized have been reported to bind Tip (58, 66). Of these proteins, there may be interesting candidates interfering with cellular signaling pathways contributing to transformation. Further investigations using other defined Tip mutants in the viral context will help to identify required interaction domains of Tip with known and unknown partners and will define which pathways modulated by Tip are required for HVS-mediated T-cell transformation.
ACKNOWLEDGMENTS
We thank Jens-Christian Albrecht for critically reading the manuscript.
This project was supported in part by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 466 project C8), the Interdisciplinary Center for Clinical Research (IZKF: Genesis, Diagnostics and Therapy of Inflammation Processes) at the University of Erlangen-Nuremberg supported by the Federal Ministry of Education and Research (BMBF FKZ 01 KS 9601), and the Wilhelm Sander-Stiftung (2002.033.1).
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ABSTRACT
Herpesvirus saimiri (saimirine herpesvirus 2) (HVS), a T-lymphotropic tumor virus, induces lymphoproliferative disease in several species of New World primates. In addition, strains of HVS subgroup C are able to transform T cells of Old World primates, including humans, to permanently growing T-cell lines. In concert with the Stp oncoprotein, the tyrosine kinase-interacting protein (Tip) of HVS C488 is required for T-cell transformation in vitro and lymphoma induction in vivo. Tip was previously shown to interact with the protein tyrosine kinase Lck. Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis and has also been detected in HVS-transformed T-cell lines. Furthermore, Tip contains a putative consensus YXPQ binding motif for the SH2 (src homology 2) domains of STAT1 and STAT3. Tip tyrosine phosphorylation at this site was required for binding of STATs and induction of STAT-dependent transcription. Here we sought to address the relevance of STAT activation for transformation of human T cells by introducing a tyrosine-to-phenylalanine mutation in the YXPQ motif of Tip of HVS C488. Unexpectedly, the recombinant virus was still able to transform human T lymphocytes, but it had lost its capability to activate STAT3 as well as STAT1. This demonstrates that growth transformation by HVS is independent of STAT3 activation.
INTRODUCTION
Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that were originally discovered as key mediators of cytokine signaling. They are involved in signaling by the interleukin-6 (IL-6) family cytokines, the IL-2 family cytokines, and numerous growth factors, including epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor 1. Latent inactive STATs reside in the cytoplasm; in response to receptor activation, they are recruited to a receptor where they are tyrosine phosphorylated by receptor tyrosine kinases (RTK) or receptor-associated tyrosine kinases, such as JAK or Trk kinases (reviewed in references 36, 42, 46, and 48). Active STAT dimers are then formed via the reciprocal interaction between the SH2 domain of one monomer and the phosphorylated tyrosine of the other. Activated dimers translocate to the nucleus, where they bind to specific DNA response elements in the promoters of target genes and activate transcription. Furthermore, various oncoproteins can activate specific STAT molecules (especially STAT3 and STAT5) (8, 15), and inappropriate STAT activation directly contributes to oncogenesis by stimulating cell proliferation and preventing apoptosis. Constitutive activation of several STATs was frequently detected in a wide range of human cell lines and primary tumors, including lymphoid malignancies (lymphomas, leukemias, and multiple myelomas) (30, 63, 67). Constitutively activated mutant STAT3 and STAT5 proteins have been shown to possess transforming properties and to be strongly associated with tumor development and progression (7, 9).
Herpesvirus saimiri (HVS), the prototype of the rhadinoviruses (gamma-2-herpesviruses), regularly establishes lifelong persistent infections in squirrel monkeys (Saimiri sciureus) of South American rainforests. While it does not cause disease in its natural host (54), experimental infection with HVS causes acute peripheral T-cell lymphoma in other New World primate species, such as tamarins, common marmosets, or owl monkeys (29). HVS strains were classified into three subgroups (A, B, and C) according to pathogenic properties and sequence variation in the left-terminal nonrepetitive genomic region (18, 52). Certain subgroup C strains are able to stimulate human T lymphocytes to stable antigen-independent growth in culture (4).
The genetic determinants responsible for induction of T-cell leukemia and T-cell transformation in vitro are located in the variable region at the left end of the HVS low GC content DNA (13, 17, 19, 21, 45, 55). At this position, virus strains of subgroup C have two open reading frames, termed stpC (saimiri transformation-associated protein of subgroup C strains) and tip (tyrosine kinase-interacting protein), which are transcribed into a single bicistronic mRNA (5, 6, 25, 38). While not required for viral replication, deletion of either stpC or tip abolishes transformation by HVS in vitro and pathogenicity in vivo (21, 22, 44, 51). StpC was shown to interact with the small G-protein Ras and stimulated mitogen-activated protein kinase activity (39). StpC further interacts with tumor necrosis factor receptor-associated factors, leading to nuclear factor kappa B (NF-B) activation (47).
Tip was identified as a 40-kDa phosphoprotein that coprecipitated with the T-cell-specific nonreceptor tyrosine kinase p56lck from lysates of C488-transformed T cells (6). The Tip kinase-interacting domain consists of two motifs, nine amino acids with homology to the C-terminal regulatory regions of various Src kinases (CSKH) and a proline-rich SH3 domain-binding sequence (SH3B), both of which are required for the interaction with the kinase (2, 6, 40). Tip is a substrate for the tyrosine kinase Lck (6, 40) and contains several tyrosine residues, three of which are conserved in all virus strains investigated (24, 31). Tip binding to Lck was reported to activate the enzyme (26, 34, 49, 64). On the other hand, in fibroblasts transformed with a constitutively active mutant of Lck, overexpression of Tip of strain C488 reversed the transformed phenotype. This was taken as evidence that Tip can downmodulate Lck-dependent signaling. These effects were even more pronounced when Tip Y114 was mutated to serine (Y114S) (32, 41).
In addition to Lck, coprecipitation of phosphorylated STAT1 and STAT3 with Lck and Tip-C484 was reported (50). A YXPQ motif of Tip (Y72; equivalent to Y114 of C488) conforms to a putative consensus binding site for STAT factors (62). Tyrosine 72 phosphorylation was required for STAT binding and transcription activation (33). This has also been demonstrated for the equivalent Y114 of strain C488 (43).
The published data were generated by overexpressing Tip in epithelial cells or T-cell lines, but the role of STAT activation by Tip in T-cell growth transformation has remained unclear. In the course of identifying the Tip elements required for transformation of human T cells, we addressed the question of whether constitutive activation of STAT3 plays a decisive role. A mutation of tyrosine 114 was introduced into recombinant viruses. This allowed us to study the behavior of Tip in the viral background, specifically to investigate the transforming potential on human cord blood lymphocytes (CBL). Surprisingly, a mutation of tyrosine 114 to phenylalanine (Y114F) did not abrogate the transforming ability of HVS and resulted in cell lines devoid of detectable levels of phosphorylated STAT3.
MATERIALS AND METHODS
Generation of recombinant viruses. The HVS strain C488 genome (18, 24) has been cloned into a set of overlapping cosmid clones; recombinant viruses can be reconstituted by cotransfection of overlapping cosmids into permissive owl monkey kidney (OMK) cells (23). To introduce the Y114F mutation into the viral genome, a 5' fragment of tip was PCR amplified from the left terminal cosmid 331 with primers 488stuY114F (5'-AAAGGTACCTCGAGCCTAGGCCTGAATGTGCTAGTTTCATTG-3'), which contained KpnI, XhoI, and StuI sites and 170326 (5'-GTAGTAAACTAAGAGCAAAGCAAGC-3'). The resulting fragment was digested with KpnI-SpeI and ligated into KpnI-SpeI-digested plasmid pSTBlueStpCHN (HVS C488 genome nucleotides 1463 to 2419) to obtain plasmid pSTBlueStpCY114FHN, which contains stpC and the first 338 bp of tip. A XhoI-StuI fragment of a Tip expression plasmid (40) was inserted into plasmid pSTBlueStpCY114FHN, resulting in plasmid pSTBlueStpCTipY114F. Correct insertion was verified by restriction mapping and DNA sequencing. A 1.7-kb Bst1107I fragment (HVS C488 nucleotides 721 to 2416) of pSTBlueStpCTipY114F was reinserted into Bst1107I-digested cosmid 331Bst1107I, resulting in cosmid 331-Y114F. Correct insertion into the cosmid was verified by restriction enzyme mapping. Recombinant viruses were generated by liposome-mediated cotransfection (Lipofectamine; Invitrogen) of a set of overlapping cosmids (23), including the altered cosmid 331-Y114F, into permissive OMK cells. The cosmids were linearized by restriction with NotI before transfection, which also removed the pWE15 cloning vector. The identity and purity of each recombinant virus were confirmed by PCR analysis. The correct tip sequence was verified by DNA sequencing of the complete tip gene with primers 110857/Tip-c3 (5'-CTCAGGCATCTTTCTTTGCATTTC-3') and 110858/Tip-c5 (5'-GGTGAATCACAAAACAGCACAAAC-3').
Cell culture and lymphocyte transformation. OMK cells (ATCC CRL1556) were used for propagation of HVS. The cells were cultivated in Dulbecco's modified Eagle's medium supplemented with glutamine (350 μg/ml), gentamicin (100 μg/ml), and 10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany). Virus stocks were generated by infection of confluent OMK cells in tissue culture flasks at a low multiplicity of infection. When lysis was complete, cell-free supernatants were stored at –80°C.
Lymphocyte cultures were grown in RPMI 1640 (Invitrogen, Karlsruhe, Germany) and Panserin 401 medium mixed at a ratio of 1:1 supplemented with 10% irradiated fetal calf serum (Pan Biotech, Aidenbach, Germany), glutamine (350 μg/ml), and gentamicin (100 μg/ml).
Human CBL were isolated by selective sedimentation of erythrocytes for 45 min at 37°C in 5% dextran (molecular weight of 250,000) in 150 mM NaCl. These primary cells were stimulated with 1 μg/ml phytohemagglutinin, and exogenous IL-2 (Roche Diagnostics, Mannheim, Germany) (10 units/ml) was added to the cells after 24 h. On the next day, the cells were infected as described previously (27). Five days after infection, cells were split into two cultures, and exogenous IL-2 was depleted from one of the cultures by centrifuging and washing the cells. Cell culture densities were determined by automated cell counting (Micro Cell Counter F-300 [Sysmex, Norderstedt, Germany] and Beckman-Coulter Z2 [Beckman-Coulter, Krefeld, Germany]). Transformation of the resulting T-cell lines was assessed microscopically and by observation of accelerated growth compared with uninfected control cultures, which ceased growing after 4 to 6 weeks. The transformed T-cell lines were analyzed by PCR and sequencing in order to confirm the specific viral genotype in the cells. None of the uninfected controls yielded a transformed T-cell line.
RNA isolation, cDNA synthesis, and amplification. Total cellular RNA was isolated by acidic phenol guanidinium thiocyanate-phenol-chloroform extraction (12). RNA (1.5 μg) was digested in a volume of 12 μl with 10 U of RNase-free DNase I (Roche Diagnostics) in the presence of 1 U of RNaseOUT RNase inhibitor (Invitrogen) and 1 mM dithiothreitol at 37°C for 30 min, followed by a heat inactivation step of 10 min at 70°C. The samples were then divided into two parallel reaction mixtures and processed with the ThermoScript reverse transcription PCR (RT-PCR) system (Invitrogen) in 20-μl reaction mixtures with or without reverse transcriptase according to the supplier's protocol. RNA complementary to the cDNA was removed by addition of 1 U of RNase H (MBI Fermentas) and incubation for 20 min at 37°C. The reaction mixtures were stored at –20°C. A 2-μl sample of the reaction mixtures was used for RT-PCR analysis. PCR conditions were as follows: a 2-min initial denaturation step at 96°C; 39 cycles, with 1 cycle consisting of 10 s at 96°C, 20 s at 62°C, and 40 s at 70°C; a final extension step of 2 min at 70°C; and a 12°C hold. Primers were specific for HVS C488 Tip (TipL, 5'-ATCCCATGTTGCTGACAAGTCACG-3', and TipR, 5'-CAAACACGTCAAGCAGTAGTGGCAG-3') and for -actin (hActin5, 5'-CCAAGGCCAACCGCGAGAAGATGAC-3', and hActin3, 5'-AGGGTACATGGTGGTGCCGCCAGAC-3').
DNA sequence analysis. Nucleotide sequences were determined with an ABI PRISM 3100 genetic analyzer (Applied Biosystems) using the Dye-Deoxy Terminator Sequencing kit according to the manufacturer's instructions (Perkin-Elmer). DNA sequence evaluation was performed with the GAP4 software (Staden Package) (16).
Immunoblotting. Cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris-HCl [pH 8], 150 mM NaCl, 1% NP-40, sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with 1 mM sodium orthovanadate (Na3VO4), 5 mM NaF, 10 μg/ml of aprotinin, and 10 μg/ml of leupeptin (Sigma-Aldrich, Taufkirchen, Germany). Total protein concentration was determined by the bicinchoninic acid assay (Pierce, Rockford, IL). Cell lysates (20 μg per lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, Mass.). Membranes were incubated at room temperature for 1 h or at 4°C overnight in blocking buffer (phosphate-buffered saline, pH 7.4, 0.1% Tween 20, 5% [wt/vol] nonfat dried milk powder) and then incubated with primary antibody diluted in blocking buffer for 1 hour or overnight. Equal loading and electrotransfer were controlled by Coomassie brilliant blue R-250 (CBB) staining of PVDF membranes after detection of enhanced chemiluminescence. Antibodies against STAT3 (F-2) and phosphorylated-STAT3-Y705 (B-7) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a dilution of 1:1,000. Primary antibodies were detected with murine immunoglobulin-specific, horseradish peroxidase-coupled secondary antibodies (Dako, Hamburg, Germany) at a dilution of 1:1,000 and an enhanced chemiluminescence substrate (Amersham, Freiburg, Germany) by a Fuji LAS-1000 chemiluminescence detection system (Raytest, Straubenhardt, Germany).
STAT activation assay. Activation of STAT1 and STAT3 was determined using a TransAM STAT family transcription factor assay (Active Motif, Carlsbad, CA) according to the manufacturer's recommendations. Cell nuclei were prepared from transformed lymphocytes by hypotonic lysis. Nuclear lysates (8 μg of total protein) were incubated in 96-well dishes containing immobilized oligonucleotides containing a STAT consensus DNA-binding site (5'-TTCCCGGAA-3') for 1 h at room temperature. Wells were then washed three times, and 100 μl of monoclonal antibody (1:1,000 dilution) was added to each well for 1 h at room temperature. Wells were washed three times, and then 100 μl of horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution) was added to each well for 1 h at room temperature. Wells were washed four times, and 100 μl of developing solution was added to each well for 10 min at room temperature. Stop solution (100 μl) was added to each well, and the absorbance at 450 nm was determined using an enzyme-linked immunosorbent assay (ELISA) reader set to 450 nm. Specificity of binding was determined by competition with wild-type and mutated STAT oligonucleotides. Nuclear extracts from HepG2 cells (treated with IL-6, 100 ng/ml) were included as positive controls for STAT3 and nuclear extracts from U-937 cells (treated with tetradecanoyl phorbol acetate and gamma interferon) for STAT1.
Flow cytometry. Transformed human T cells were analyzed by flow cytometry with antibodies for T-cell surface epitopes on a FACScalibur flow cytometer (Becton Dickinson) according to standard protocols. The directly labeled monoclonal antibodies (Cy-Chrome, fluorescein isothiocyanate, or phycoerythrin conjugated) were specific against CD3 (Leu-4), CD4 (Leu-3a), CD8 (Leu-2a), CD25 (M-A251), CD28 (L293), HLA-DR (L243), CD80 (L307.4), and CD86 (IT2.2) (all from BD Biosciences Immunocytometry Systems and Pharmingen). Directly labeled isotype-matched control monoclonal antibodies were used (BD Biosciences Immunocytometry Systems and Pharmingen).
RESULTS
Construction of recombinant viruses. In order to identify the relevant motifs of the Tip protein required for transformation of human T lymphocytes by HVS, we set out to construct recombinant viruses carrying defined mutations in the tip coding sequence. First, we addressed the relevance of STAT factors by mutating the Tip residue responsible for STAT activation. A shuttle plasmid containing the stpC and tip genes was generated, and mutations were introduced by standard cloning and PCR-based mutagenesis. After verification by DNA sequencing, the mutated tip was introduced into a cosmid encompassing the left terminal region of HVS strain C488. In more detail, the respective stpC and tip genes were subcloned into plasmid pSTBlue and the tyrosine residue 114 was replaced by phenylalanine (Y114F). The modified coding sequence was then reinserted into the left terminal cosmid 331Bst1107I, which lacks the stpC and tip genes, to generate cosmid 331-Y114F (Fig. 1). Recombinant virus was then generated by homologous recombination of five overlapping cosmids in permissive OMK cells, which allows construction of recombinant viruses without wild-type virus contamination (23). The viruses M11 (wild-type C488) and two independent recombinant C488-Y114F obtained after cotransfection were verified by PCR, and the tip gene was sequenced.
Transformation of human T cells in vitro. Herpesvirus saimiri C488 is able to transform human T cells to antigen-independent growth in vitro (4). In order to test the transforming potential on human CBL, the CBL were infected in parallel with wild-type C488 or cosmid-generated wild-type M11 control viruses and recombinant Tip-mutated viruses. Proliferation of the cells was compared to that of uninfected controls. Cells were regarded as growth-transformed cells when they had proliferated continuously for at least 3 months while uninfected control cells had ceased growing. Transformed T-cell lines were analyzed by PCR and sequencing in order to confirm the specific viral genotype in the cells.
The uninfected control cells stopped growing after 2 to 3 weeks of culture. Surprisingly, the HVS C488-Y114F mutant was still capable of transforming human T cells to antigen-independent growth in the presence or absence of exogenous IL-2. The experiments were performed with two independently generated C488-Y114F mutants; both recombinants yielded identical results. Moreover, the cells whose growth had been transformed with Tip-Y114F recombinant viruses grew to higher cell numbers than the wild-type HVS C488-transformed cells (Fig. 2 and 3). Thus, growth transformation of human T lymphocytes by HVS is independent of the presence of the tyrosine residue at position 114.
Altered morphology and phenotype of transformed cells. The surface phenotypes of the human CBL-derived T-cell lines successfully transformed with wild-type/M11 and C488-Y114F viruses were analyzed by flow cytometry. Herpesvirus saimiri-transformed T lymphocytes show the surface markers characteristic of mature, activated T cells (4, 27, 28, 53). Figure 4 shows the surface marker expression of HVS C488- and HVS C488-Y114F-transformed cells. Wild-type/M11 and C488-Y114F-transformed cells showed no differences in CD3 and HLA-DR expression. Y114F-transformed cells had a high expression level of CD25, regardless of the presence or absence of exogenous IL-2, and they show a higher CD28 expression compared to wild-type virus-transformed cells. With regard to CD28 ligands, all wild-type virus-transformed cells tested were positive for CD80 (B-7.1) only, whereas C488-Y114F-transformed cell lines expressed both CD80 and CD86 (B-7.2) activation markers. While cultures growth transformed by HVS C488 or M11 wild-type virus consisted of CD4+ cells or CD8+ cells, Tip Y114F-transformed CBL had a CD4+ phenotype; a minor fraction of CD4+ CD8+cells was found in some of the cell lines.
The expression level of Tip could theoretically contribute to these differences, although expression of HVS stpC/tip oncogenes has been found to be highly variable in successfully transformed human T cells (25, 26). As Tip expression in transformed cell lines is hardly detectable by Western blotting, we used RT-PCR to analyze the transcription of tip in T-cell lines transformed by wild-type and Y114F viruses. RT-PCR for tip and -actin was performed on total RNA with appropriate controls, and reaction products were separated by agarose gel electrophoresis. A 413-bp fragment specific for tip (Fig. 5) was detected in all infected cultures. RT-PCR for -actin (587-bp fragment) shows that similar amounts of cellular RNA were used for cDNA synthesis (Fig. 5). According to the result of this experiment, tip transcription is variable, but no consistent difference was seen.
Phosphorylation and activation status of STAT3. Interaction of Tip with Lck and STAT has been investigated for HVS strain C484 in vitro and in transfected cells: phosphorylated STAT1 and STAT3 were immunoprecipitated together with Tip-C484 and Lck from lysates of transfected Jurkat cells (49). It has further been shown that Tip-C484 enhances the activity of STAT1 and STAT3 in the presence of Lck. For activation of STAT-dependent transcription, tyrosine 72 of Tip (identical to Y114 of C488), which is part of a putative consensus binding site (YXPQ) for STAT factors, was required (33).
The phosphorylation of STAT3 in HVS C488-Y114F-transformed cell lines was compared to wild-type HVS-transformed T cells by Western blotting. As to be expected from earlier reports, Y705 of STAT3 was found to be tyrosine phosphorylated in wild-type virus-transformed human T cells. No significant phosphorylation at the regulatory Y705 was detected in HVS C488-Y114F-transformed human T cells, while STAT3 was expressed at approximately equal levels in all cell lines tested (Fig. 6A). In immunoblots with two different Y701 phosphorylation-specific antibodies, STAT1 phosphorylation at the regulatory Y701 was detected only weakly or was absent in wild-type-virus-transformed T cells, and Y114F-transformed cells showed no noticeable STAT1 Y701 phosphorylation (data not shown). In addition, binding of STAT1 and STAT3 to specific DNA recognition sites was analyzed by an ELISA-based assay that allows detection and quantification of DNA-binding activity of transcription factors (Fig. 6B). This assay demonstrated that HVS C488-transformed cells show a weak but specific STAT1 activation, whereas Y114F-transformed T-cell lines show no activation of STAT1. The STAT3 assay showed strong activation in HVS C488-transformed cells, while Y114F cell lines exhibited no STAT3-specific DNA binding and thus no activation at all. The specificity of the assay is monitored by addition of a wild-type consensus oligonucleotide which acts as a competitor. In summary, both tyrosine phosphorylation and DNA-binding activity of STAT1 and STAT3, which were regularly found in wild-type virus-transformed T cells, was not observed in human T cells growth transformed by HVS C488-Y114F.
DISCUSSION
HVS causes rapidly progressive T-cell leukemia and lymphoma in several species of New World primates, while it is not pathogenic in its persistently infected natural host, the squirrel monkey (Saimiri sciureus) (29). Strains of the highly oncogenic subgroup C, particularly strain C488, are also capable of transforming primary human T cells to permanent antigen-independent growth (4). Two open reading frames, stpC and tip, are necessary for the transforming and pathogenic properties of HVS C488 (1, 20).
So far, the essential contribution of Tip to T-cell transformation by HVS was mainly attributed to an Lck-mediated STAT3 activation. This hypothesis was based on the constitutive STAT3 activation found in transformed human and monkey T cells as a consequence of transformation by HVS of subgroup C and also of subgroup A (14, 59, 61), as well as in human cell lines cotransfected with Tip- and Lck-encoding plasmids (33, 43, 50). It was supported by numerous studies showing that improper STAT activation contributes to oncogenesis by stimulation of cell proliferation and inhibition of apoptosis. Moreover, a variety of human cell lines and primary tumors exhibits constitutively active STATs (3, 10, 30, 37, 56, 63, 65, 67).
For Tip of HVS strain C484, it had been shown that phosphorylation of tyrosine 72, which is part of a putative consensus binding site for STAT factors (YXPQ), is required for STAT binding and transcriptional activation (33). The YXPQ interaction motif of Tip is well conserved among different isolates of HVS subgroup C (24, 31). Accordingly, Y114 of Tip-C488, like Y72 of Tip-C484, is required for stimulation of STAT-dependent reporter gene transcription in transiently transfected Jurkat T cells, and mutation of the respective residue results in loss of STAT3 activation (33, 43).
To challenge the hypothesis of an essential function for STAT3 activation in human T-cell transformation by HVS strain C488, we inserted a Tip-Y114F mutation into recombinant viruses. Our data show that two independently generated recombinant HVS C488-Y114F viruses were still able to transform human T cells to antigen-independent growth in culture (Fig. 3). At the same time, constitutive STAT3 phosphorylation and activation observed in wild-type HVS C488 or recombinant cosmid-reconstituted wild-type-virus-transformed cells (Fig. 6) was lost in HVS C488-Y114F-transformed cells. Thus, activation of STAT3 seems to play a minor role in growth transformation of human T cells by HVS.
The question arises whether the process of viral T-cell transformation is completely independent of STAT3 or whether other (STAT) factors can compensate for the missing STAT3 activation. However, STAT1 activation could not be detected in HVS C488-Y114F-transformed cells (Fig. 6B). A contribution of STAT heterodimers, which have been reported for STAT1 and STAT2 heterodimers and STAT1 and STAT3 heterodimers (reviewed in references 35, 46, and 60), is also unlikely, since neither activated STAT1 nor STAT3 was detected in nuclear extracts from HVS C488-Y114F-transformed cells (Fig. 6B).
The phenotype of lymphocytes transformed with the HVS Y114F mutant viruses was slightly different from that of cells carrying wild-type HVS. They grew to higher cell numbers and formed larger aggregates (Fig. 2 and 3). Like T cells transformed with the parental HVS C488 (28), they displayed typical surface markers of mature T cells. It has been reported that overexpression of Tip in T-cell lines results in a downmodulation of CD3 and CD4 surface expression (57), which was more pronounced in Tip-Y114S (32, 58). In our hands, HVS-transformed cells expressing the HVS oncogene tip from the viral promoter do not show such a dramatic effect; instead, they showed only a modestly lower surface expression of CD3 in C488-Y114F-transformed cells compared to HVS-C488-transformed cells and normal expression of CD4 (Fig. 4).
Surface expression of CD25 (IL-2 receptor ) and CD28 were augmented, and in addition to CD80, HVS C488-Y114F-transformed cells displayed the alternative CD28 ligand, CD86. A role of STAT3 in the regulation of these surface markers has not been described so far. The differences in the phenotype might be related to targeting of distinct T-cell populations by wild-type and mutant viruses, respectively. Alternatively, the phenotypic variation could be attributed to differentiation into divergent T-cell subpopulations in the course of transformation, which might also be influenced by STAT3 activity.
In summary, this work provides strong evidence that STAT3 (and STAT1) activation, though consistently found in T-cell lines transformed by HVS strains in vitro or in vivo (14, 59, 61), is not a prerequisite for human primary T-cell transformation to antigen-independent growth by HVS subgroup C strains. Therefore, the essential role of Tip in viral T-cell transformation has to be mediated by effectors other than STAT3. The association of Tip and Lck with the endosomal targeting protein p80 was found to responsible for downregulation of TCR and CD4 surface expression and thereby their signal transduction pathways (57). This seems to be STAT3 independent, since it was found to be even more pronounced with Tip-Y114F. Although we do not see a dramatic TCR complex modulation in transformed human T cells, pathways linked to the endocytic process might be relevant for transformation of T cells by HVS subgroup C strains. However, in general, growth regulatory mechanisms related to receptor endocytosis have not been described yet.
Therefore, we would like to suggest another model of Tip's role in T-cell transformation which largely relies on direct effects of Tip on Lck function. While activation of Lck's in vitro activity upon Tip binding has been reported repeatedly (26, 34, 43, 49, 64), several in vivo approaches argue in favor of kinase inhibition by Tip (32, 41). In a recent publication, Tip-mediated inhibition of T-cell receptor signal transduction was shown to occur at the level of ZAP70 phosphorylation and resulted in a failure to induce intracellular calcium mobilization, while phosphorylation of the T-cell receptor -chain was not affected (11). An attractive hypothesis deduced from these data would include an altered substrate specificity of Lck in the presence of Tip. Thereby, Lck activity may be redirected towards a target not yet identified. Tip may again be involved by recruiting this alternative target molecule to the Lck complex. STAT3 would have been an attractive candidate, but it is excluded by this study. Numerous other proteins not yet characterized have been reported to bind Tip (58, 66). Of these proteins, there may be interesting candidates interfering with cellular signaling pathways contributing to transformation. Further investigations using other defined Tip mutants in the viral context will help to identify required interaction domains of Tip with known and unknown partners and will define which pathways modulated by Tip are required for HVS-mediated T-cell transformation.
ACKNOWLEDGMENTS
We thank Jens-Christian Albrecht for critically reading the manuscript.
This project was supported in part by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 466 project C8), the Interdisciplinary Center for Clinical Research (IZKF: Genesis, Diagnostics and Therapy of Inflammation Processes) at the University of Erlangen-Nuremberg supported by the Federal Ministry of Education and Research (BMBF FKZ 01 KS 9601), and the Wilhelm Sander-Stiftung (2002.033.1).
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