The structure and stability of an HLA-A*0201/octameric tax peptide complex with an empty conserved peptide-N-terminal binding site.
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The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 A) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, DeltaTm = 16 degrees C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a "closed conformation" of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes. (+info)
Up-regulation of human T lymphotropic virus type 1 (HTLV-1) tax/rex mRNA in infected lung tissues.
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HTLV-1 has been implicated in certain pulmonary diseases. We previously demonstrated that expression of HTLV-1 tax/rex mRNA, encoding the transcriptional transactivator Tax, was closely associated with infiltration of activated T lymphocytes into lung tissue. To explore mechanisms of tax/rex expression in the lung, tax/rex mRNA expression and proviral DNA load were compared between peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) from four patients with HTLV-1-associated myelopathy (HAM/TSP) and 13 carriers with various pulmonary symptoms. Semiquantitative detection of tax/rex mRNA strongly suggested that the lung was a preferential site for its expression. Proviral DNA loads in non-HAM/TSP carriers were variable but correlated well between PBMC and BALC in each individual, and revealed no relationship with tax/rex mRNA expression. In contrast, both cell groups from four HAM/TSP patients expressed detectable tax/rex mRNA accompanied by high proviral DNA load. The ratio of tax/rex mRNA expression to proviral DNA load was higher in BALC than in PBMC in three of four carriers and in three of four HAM/TSP patients, suggesting up-regulation of tax/rex mRNA in infected lung tissue. To analyse differences in distribution of HTLV-1 quasispecies between the two tissues, phylogenetic analysis was performed for sequence sets of the proviral tax open reading frame (ORF: 1059 bp) derived from PBMC and BALC of two infected individuals. Sequences derived from the two tissues distributed similarly to branches of phylogenetic trees, and there was no evidence of selective distribution of certain quasispecies in the lung. Our results suggest the presence of tissue-specific conditions that activate viral expression in infected cells in the lung. Constitutive exposure of this tissue to foreign antigens leading to up-regulation of basal viral promoter activity is likely to be one such mechanism. (+info)
Constitutive activation of transcription factor AP-1 in primary adult T-cell leukemia cells.
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Human T-cell leukemia virus type-I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL). This study examined the status of the oncogenic transcription factor AP-1 in leukemic cells freshly isolated from patients with ATL. Leukemic cells from peripheral blood of all patients with ATL exhibited constitutive AP-1 DNA binding activity, whereas mononuclear cells from normal individuals did not. In agreement with previous studies, HTLV-I transforming protein, Tax, was found to stimulate the DNA binding activity of AP-1 in a T-cell line. However, HTLV-I genes, including Tax, were not significantly expressed in leukemic cells freshly obtained from patients with ATL. Moreover, all T-cell lines derived from leukemic cells of patients with ATL also displayed constitutive AP-1 DNA binding activity, but expressed little Tax protein. Thus, leukemic cells of patients with ATL appear to have Tax-independent mechanisms that induce AP-1 activity, both in vivo and in vitro. In antibody supershift experiments, AP-1 in fresh leukemic cells and ATL-derived cell lines were found to contain JunD. Consistently, all primary ATL cells and ATL-derived cell lines expressed high levels of JunD messenger RNA. Our results suggest that AP-1 is activated in leukemic cells of patients with ATL through a Tax-independent mechanism and this may play a role in the deregulated phenotypes of ATL leukemic cells. (Blood. 2000;95:3915-3921) (+info)
The HTLV-I tax protein transcriptionally modulates OX40 antigen expression.
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OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells. (+info)
Differences in the ability of human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 tax to inhibit p53 function.
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We have analyzed the functional activity of the p53 tumor suppressor in human T-cell lymphotropic virus type 2 (HTLV-2)-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, both in transient-transfection assays using a p53 reporter plasmid and in induction of p53-responsive genes in response to gamma irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. In transient-cotransfection assays, Tax-1 and Tax-2B inactivated p53 by 80%, while Tax2A reduced p53 activity by 20%. In addition, Tax-2A does not increase the steady-state level of cellular p53 as well as Tax-1 or -2B does in the same assays. Cotransfection assays demonstrated that Tax-2A could efficiently transactivate CREB-responsive promoters to the same level as Tax-1 and Tax-2B, indicating that the protein was functional. This report provides evidence of the first functional difference between the HTLV-2A and -2B subtypes. This comparison of the action of HTLV-1 and HTLV-2 Tax proteins on p53 function will provide important insights into the mechanism of HTLV transformation. (+info)
Bcl-X(L) is up-regulated by HTLV-I and HTLV-II in vitro and in ex vivo ATLL samples.
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Human T lymphotropic virus type I (HTLV-I) is the etiological agent of adult T-cell lymphocytic leukemia (ATLL), whereas HTLV-II has not been associated with hematopoietic malignancies. The control of apoptotic pathways has emerged as a critical step in the development of many cancer types. As a result, the underlying mechanism of long-term survival of HTLV-I and HTLV-II was studied in infected T cells in vitro and in ex vivo ATLL samples. Results indicate that HTLV-I- and HTLV-II-infected T cells in vitro express high levels of the antiapoptotic protein Bcl compared with other human leukemic T cell lines or uninfected peripheral blood mononuclear cells. The levels of proapoptotic proteins Bax, BAD, and Bak were not significantly altered. HTLV-I and HTLV-II viral transactivators, Tax1 and Tax2, are known to increase expression of cellular genes. These proteins were tested for increased transcription from the human Bcl2 and Bcl-X(L) promoters. Whereas no effect was observed on the Bcl2 promoter, both Tax1 and Tax2 increased transcription of the Bcl-X(L) promoter in T cells, although Tax1 appeared to be more efficient than Tax2. The biological significance of these observations was validated by the finding of an increased expression of Bcl-X(L) in ex vivo ATLL cells, especially from patients unresponsive to various chemotherapy regimens. Altogether, these data suggest that overexpression of Bcl-X(L )in vivo( )may be in part responsible for the resistance of ATLL cells to chemotherapy. In addition, inefficient activation of the Bcl-X(L) promoter by Tax2 may result in a shorter survival time of HTLV-II-infected cells in vivo and a diminished risk of leukemia development. (+info)
Domain-specific interaction with the I kappa B kinase (IKK)regulatory subunit IKK gamma is an essential step in tax-mediated activation of IKK.
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The human T-cell leukemia virus type 1 Tax oncoprotein deregulates the NF-kappa B signaling pathway by persistently stimulating a key signal transducer, the I kappa B kinase (IKK). Tax physically associates with the IKK regulatory subunit, IKK gamma, although the underlying biochemical mechanism and functional significance remain unclear. We show that the Tax-IKK gamma interaction requires two homologous leucine zipper domains located within IKK gamma. These leucine zipper domains are unique for the presence of a conserved upstream region that is essential for Tax binding. Site-directed mutagenesis analysis revealed that a leucine-repeat region of Tax is important for IKK gamma binding. Interestingly, all the Tax mutants defective in IKK gamma binding failed to engage the IKK complex or stimulate IKK activity, and these functional defects can be rescued by fusing the Tax mutants to IKK gamma. These results provide mechanistic insights into how Tax specifically targets and functionally activates the cellular kinase IKK. (+info)
Overexpression of p21(waf1) in human T-cell lymphotropic virus type 1-infected cells and its association with cyclin A/cdk2.
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Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G(1), S, or G(2)/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G(1) phase prior to gamma-irradiation, arrested in G(1) and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G(1)/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry. (+info)