Human T-cell Leukemia Virus Tax Research Articles

Akt activation by human T-cell leukemia virus tax oncoprotein

Human T-Cell Leukemia Virus type 1 (HTLV-1), the etiological agent of Adult T-Cell Leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ (post synaptic density protein) domain binding motif (PBM), previously reported to be important for non-canonical nuclear factor kappa B (NFkappaB) activation. In contrast, the current study finds no defect in non-canonical NFkappaB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt (Protein Kinase B) activation. Tax1 attenuated the effects of negative regulatory phosphatases of the PI3K-Akt-mTOR pathway, PTEN (Phosphatase and Tensin homologue) and PHLPP (PH domain and leucine rich repeat protein phosphatase). Tax1 competes with PTEN for binding to PDZ protein DLG-1 (Drosophila disc large tumor suppressor), unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP, by fusion to a myristolyation acceptor motif, overcame the effects of Tax1, as measured by levels of Akt phosphorylation at T308 and S473, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2. Moreover, these findings suggest a new approach to therapeutics for HTLV-1 lymphoproliferative disease. Supported by NIH grants CA94056, CA1730, CA63413, Lymphoma Leukemia Society grant 6067-10, American Society for Hematology fellowship and NIH T32 grant HL07088, and Lymphoma Research Foundation grant 307181203.

Activation of AID by human T-cell leukemia virus Tax oncoprotein and the possible role of its constitutive expression in ATL genesis

Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1). Mutations of tumor suppressor genes have been described in ATL. Although Tax, a product of HTLV-1, is associated with cellular genetic aberrations, the mechanisms of such association are not fully clear. Activation-induced cytidine deaminase (AID) is involved in somatic DNA alterations of the immunoglobulin gene for amplification of immune diversity. However, inappropriate expression of AID acts as a genomic mutator that contributes to tumorigenesis. To gain insight into the molecular mechanism underlying the emergence of somatic mutations in various genes during leukemogenesis, we examined the expression of AID. HTLV-1-infected T-cell lines and ATL cells expressed high levels of AID compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Immunohistochemistry showed AID-positive ATL cells in lymph nodes and skin lesions. Infection of a human T-cell line and normal PBMCs with HTLV-1 induced AID expression. Tax transcriptionally activated AID gene through both the nuclear factor-kappaB subunit p50 and cyclic adenosine 3',5'-monophosphate response element-binding protein signaling pathways. p50, which lacks a transactivation domain, interacted with the transcriptional coactivator Bcl-3 in HTLV-1-infected T cells. Thus, activation of p50/Bcl-3 complexes in T cells in response to Tax might explain the constitutive expression of AID in HTLV-1-infected T cells. The constitutive expression of AID in ATL cells can be speculated to result from mutations induced by the Tax-activated AID and/or other Tax-associated mutagenic mechanisms during the pre-leukemic stage, which cause functional modification within the AID promoter or in any of its cellular regulatory activator proteins.

Exclusive Ubiquitination and Sumoylation on Overlapping Lysine Residues Mediate NF-κB Activation by the Human T-Cell Leukemia VirusTax Oncoprotein

The transcription factor NF-kappaB is critical for the induction of cancer, including adult T-cell leukemia, which is linked to infection by human T-cell leukemia virus type 1 and the expression of its regulatory protein Tax. Although activation of the NF-kappaB pathway by Tax involves its interaction with the regulatory subunit of the IkappaB kinase (IKK) complex, NEMO/IKKgamma, the mechanism by which Tax activates specific cellular genes in the nucleus remains unknown. Here, we demonstrate that the attachment of SUMO-1 to Tax regulates its localization in nuclear bodies and the recruitment of both the RelA subunit of NF-kappaB and free IKKgamma in these nuclear structures. However, this sumoylation step is not sufficient for the activation of the NF-kappaB pathway by Tax. This activity requires the prior ubiquitination and colocalization of ubiquitinated Tax with IKK complexes in the cytoplasm and the subsequent migration of the RelA subunit of NF-kappaB to the nucleus. Thus, the ubiquitination and sumoylation of Tax function in concert to result in the migration of RelA to the nucleus and its accumulation with IKKgamma in nuclear bodies for activation of gene expression. These modifications may result in targets for the treatment of adult T-cell leukemia.

Somatic mutagenesis studies of NF-kappa B signaling in human T cells: evidence for an essential role of IKK gamma in NF-kappa B activation by T-cell costimulatory signals and HTLV-I Tax protein.

NF-kappa B plays a pivotal role in normal T-cell activation and may also mediate human T-cell leukemia virus (HTLV)-induced T-cell transformation. Activation of NF-kappa B by both T-cell costimulatory signals and the HTLV Tax protein involves stimulation of I kappa B kinase (IKK). As a genetic approach to dissect the intermediate steps involved in NF-kappa B activation in human T cells, we performed somatic cell mutagenesis to isolate signaling-defective mutant Jurkat T-cell lines. One of the mutant cell lines was shown to have a specific blockade in the IKK signaling pathway but remained competent in the c-Jun N-terminal kinase and MAP kinase pathways. Interestingly, this mutant cell line lacks expression of IKK gamma, a non-catalytic component of the IKK complex. Expression of exogenous IKK gamma in the mutant cells restored NF-kappa B activation by both the T-cell costimulation agents and Tax. These findings provide genetic evidence for the requirement of IKK gamma in NF-kappa B signaling triggered by both T-cell costimulatory signals and HTLV-I Tax protein.

Identification of a Human T-cell Leukemia Virus Type I Tax Peptide in Contact with DNA

The human T-cell leukemia virus Tax protein directs binding of a host factor, cAMP response element binding protein, to an extended recognition sequence in the proviral promoter. Prior cross-linking experiments have revealed that Tax makes restricted contact with this DNA at two symmetric positions, 14 nucleotides apart on opposite strands of the DNA. Tax lacks a conventional DNA binding domain, and the sequences in Tax that are in contact with DNA have not been previously identified. Analysis of cross-linked peptides now shows that the contact occurs between Tax residues 89 and 110, corresponding to a protease-sensitive linker joining two protein structural domains. The linker assumes a protease-resistant conformation in the cross-linked complex. Point mutations within the linker prevent cross-linking and interfere with Tax function. These data suggest that entry of Tax into the ternary complex may be coupled to folding of an unstructured protein domain, which then makes base-specific contacts with DNA.

IKKγ Serves as a Docking Subunit of the IκB Kinase (IKK) and Mediates Interaction of IKK with the Human T-cell Leukemia Virus Tax Protein

The tax gene product of human T-cell leukemia virus type I induces activation of transcription factor NF-kappaB, which contributes to deregulated expression of various cellular genes. Tax expression triggers persistent phosphorylation and degradation of the NF-kappaB inhibitory proteins IkappaBalpha and IkappaBbeta, resulting in constitutive nuclear expression of NF-kappaB. Recent studies demonstrate that Tax activates the IkappaB kinase (IKK), although the underlying mechanism remains unclear. In this report, we show that Tax physically interacts with a regulatory component of the IKK complex, the NF-kappaB essential modulator or IKKgamma (NEMO/IKKgamma). This molecular interaction appears to be important for recruiting Tax to the IKK catalytic subunits, IKKalpha and IKKbeta. Expression of NEMO/IKKgamma greatly promotes binding of Tax to IKKalpha and IKKbeta and stimulates Tax-mediated IKK activation. Interestingly, a mutant form of Tax defective in IKK activation exhibited a markedly diminished level of NEMO/IKKgamma association. These findings suggest that the physical interaction of Tax with NEMO/IKKgamma may play an important role in Tax-mediated IKK activation.

Binding of the human T-cell leukemia virus Tax protein to the coactivator CBP interferes with CBP-mediated transcriptional control.

The HTLV-I oncoprotein Tax is required for high level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Tax stimulates HTLV-I transcription through high affinity binding to the KIX domain of CBP, a pleiotropic coactivator. Several cellular proteins, including c-jun, also bind to KIX and utilize CBP as a coactivator. To test whether Tax binding to KIX may disable cellular CBP function, we examined the potential interplay between Tax and c-jun for binding to KIX. We show that Tax represses the transcription function of c-jun in vivo and demonstrate that both transcription factors bind to an overlapping minimal region of KIX in vitro. c-jun binding to KIX is displaced by Tax, indicating that their binding is mutually exclusive and providing a molecular basis for the observed repression. The competition between Tax and cellular transcription factors for CBP represents a novel pathway for HTLV-I dependent deregulation of gene expression, and may have significant implications for cellular homeostasis and transformation in the HTLV-I infected T-cell.

Molecular interactions between the coactivator CBP and the human T-cell leukemia virus tax protein

The oncoprotein Tax, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high-level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Recent evidence suggests that Tax stimulates HTLV-I transcription through recruitment of the cellular coactivator protein CBP to the HTLV-I promoter, promoting high-level viral replication via the transcriptional activation properties associated with CBP. Tax directly contacts the KIX domain of CBP to stably anchor the coactivator to nucleoprotein complexes at the promoter. Here, we identify KIX amino acid residues 588 to 683 as the minimal region sufficient for interaction with Tax. This region is similar to the minimal KIX amino acid residues necessary for strong interaction with phosphorylated CREB, and is composed of a structural domain that forms an extensive hydrophobic core. We further show that a double point mutation in KIX differentially affects the binding of Tax and phosphorylated CREB, suggesting that these transcription factors may recognize unique amino acid residues within the KIX domain. These observations suggest that Tax directly contacts the hydrophobic core of KIX, and provides a structural framework to further define the molecular interactions between Tax and CBP.

A Human T-cell Leukemia Virus Tax Variant Incapable of Activating NF-κB Retains Its Immortalizing Potential for Primary T-lymphocytes

The human T-cell leukemia virus type 1 (HTLV-1) transactivator (Tax) has been shown to interfere with regulated cellular proliferation. Many studies have focused on the ability of Tax to transform rodent fibroblasts; however, none has defined the molecular requirements for Tax transformation of human lymphoid cells. We show here that tax induces permanent growth of human primary T-lymphocytes by using a transformation/immortalization defective rhadinovirus vector. The cells phenotypically resemble HTLV-immortalized lymphocytes and contain episomally persisting recombinant rhadinoviral sequences, which stably express functional Tax protein. As Tax can activate major cellular signal transducing pathways including NF-kappaB and cAMP-responsive element binding protein (CREB), we asked for the relevance of these routes in the immortalization of human lymphocytes. By using Tax mutants that either activate exclusively CREB/activating transcription factor or are defective in activating this signaling pathway, we delineated that Tax can induce immortalization of primary human T-lymphocytes through a mechanism independent of NF-kappaB activation.

Specific interaction of HTLV tax protein and a human type IV neuronal intermediate filament protein.

The human T-cell leukemia virus (HTLV) is associated with adult T cell leukemia and neurological disorders (TSP/HAM). The HTLV transcriptional transactivator, Tax, is known to exert its effect through protein-protein interaction with several transcription factors that activate genes in T cell proliferation. The pathogenic mechanism in the CNS is less defined. Using the yeast two-hybrid system, we have identified a specific Tax-binding protein as the neuronal specific intermediate filament protein, alpha-internexin. Tax binds to the domain corresponding to the rod region of alpha-internexin, which is essential for neurofilament assembly. The Tax domains involved in binding are separable from those involved in transactivation. TxBP-1/alpha-internexin and Tax are expressed in the cytoplasm and nucleus, respectively, when expressed alone, but in coexpressing cells, colocalization of both proteins was observed in a perinuclear, punctate distribution. This in vivo interaction also resulted in a dramatic reduction in Tax transactivation and the network formation by alpha-internexin. The specific interaction of Tax and a neuronal specific intermediate filament protein may provide a clue to the pathogenesis of TSP/HAM.

Viral transactivating proteins.

Many viruses utilize the cellular transcription apparatus to express their genomes, and they encode transcriptional regulatory proteins that modulate the process. Here we review the current understanding of three viral regulatory proteins. The adenovirus E1A protein acts within the nucleus to regulate transcription through its ability to bind to other proteins. The herpes simplex type 1 virus VP16 protein acts within the nucleus to control transcription by binding to DNA in conjunction with cellular proteins. The human T-cell leukemia virus Tax protein influences transcription through interactions with cellular proteins in the nucleus as well as the cytoplasm.

Activation of the IL-2 gene promoter by HTLV-I tax involves induction of NF-AT complexes bound to the CD28-responsive element.

The tax gene product of the type I human T-cell leukemia virus (HTLV-I) is a potent transcriptional activator of various growth-related cellular genes, including that encoding interleukin-2 (IL-2). Tax activation of many of these target genes appears to be mediated by the NF-kappa B/Rel and CREB/ATF family of cellular transcription factors. However, the mechanism by which Tax transactivates the IL-2 gene remains unclear. In the present study, we demonstrate that neither NF-kappa B/Rel nor CREB/ATF is sufficient for Tax-mediated activation of the IL-2 promoter. Two novel nuclear protein complexes are induced by Tax and specifically bind to an IL-2 gene enhancer, the CD28-responsive element (CD28RE). Immunobiochemical analyses suggest that these DNA binding complexes contain at least two members of the nuclear factor of activated T cells, NF-ATp and NF-ATc. However, the CD28 binding NF-AT complexes do not contain Jun and Fos family proteins that have been proposed to serve as NF-AT partners in the activation of the IL-2 NF-AT motif. Transient transfection studies demonstrate that the in vivo expressed NF-ATp binds to the CD28RE probe and enhances Tax-mediated activation of this critical IL-2 enhancer. We demonstrate further that binding of NF-AT to CD28RE is critical for Tax activation of the IL-2 promoter. Together, these results suggest a novel mechanism of Tax-mediated activation of the IL-2 gene, which involves the induction of NF-AT-containing CD28RE binding complexes.

Transactivation by the human T-cell leukemia virus Tax protein is mediated through enhanced binding of activating transcription factor-2 (ATF-2) ATF-2 response and cAMP element-binding protein (CREB).

The human T-cell leukemia virus type I (HTLV-I)-encoded transcriptional activator protein Tax is strongly implicated in HTLV-I pathogenesis. Tax regulates HTLV-I gene expression through three 21-base pair (bp) repeat enhancer elements located in the transcriptional control region of the virus. Tax does not bind these elements directly, but mediates transactivation through the cellular transcription factors that recognize a cAMP response element (CRE)-like sequence centered within each of the 21-bp repeats. In this report, we identify activating transcription factor-2 (ATF-2) and CRE-binding protein (CREB) as the principal T-cell proteins that bind the three 21-bp repeats in vitro. Purified Tax protein augments the level of RNA synthesis induced by ATF-2 and CREB in a cell-free transcription assay, providing evidence that Tax cooperates with these cellular proteins to activate HTLV-I transcription. Furthermore, Tax dramatically increases the binding of both the T-cell-derived and recombinant forms of ATF-2 and CREB to each of the 21-bp repeats. The target sequences for this enhancement reside within the DNA binding/dimerization domains of these proteins. These data suggest that Tax transactivates HTLV-I gene expression by increasing the number of bound ATF-2 and CREB molecules at the viral promoter.

Pleiotropic effect of the human T-cell leukemia virus Tax protein on the DNA binding activity of eukaryotic transcription factors.

The Tax protein, encoded by the human T-cell leukemia virus type I, is a potent activator of viral and cellular gene transcription. Tax does not bind DNA directly but appears to trans-activate through an interaction with host-cell transcription factors that recognize sequences within the promoters of Tax-responsive genes. Cellular transcriptional activators implicated in mediating Tax trans-activation include members of the activating transcription factor/cAMP response element binding protein (ATF/CREB) family of proteins, serum response factor, Fos-Jun, and NF-kappa B. Recent evidence suggests that Tax may stimulate human T-cell leukemia virus type I transcription, at least in part, through enhanced binding of ATF/CREB proteins to their recognition elements within the Tax-responsive 21-bp repeats of the viral promoter. In this report, we demonstrate that Tax also enhances the site-specific DNA binding activity of serum response factor and Fos-Jun and modestly enhances the binding of the NF-kappa B subunits, p50 and p65. We also show that Tax increases the DNA binding activity of the eukaryotic transcription factors ATF-1, Sp1, and GAL4. These results are consistent with the finding that Tax is highly pleiotropic and suggest that Tax trans-activation may involve enhancement in the DNA binding activity of target transcriptional regulatory proteins. In addition, we show that the mechanism of Tax-enhanced DNA binding activity does not involve an alteration in the redox state of the target protein.

The indirect association of human T-cell leukemia virus tax protein with DNA results in transcriptional activation

trans-activator p40tax of human T-cell leukemia virus type I activates specific enhancers and stimulates transcription of the viral and some cellular genes but does not bind directly to the enhancer sequences. We demonstrated here that a fusion protein of p40tax and GAL4 DNA binding domain activated transcription dependent on the GAL4-binding site in the reporter plasmid. Mutants of p40tax were also tested in the fusion protein, and their activities were found to be in parallel with those of their free forms on the original target long terminal repeat. This activation with GAL4 fusion protein was interfered with by the free form of p40tax. These results suggest that p40tax associates with DNA through interaction with DNA binding protein(s) and also interacts with another transcription factor(s) to elicit the activation.

Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes.

The type I human T-cell leukemia virus (HTLV-I) encodes a 40-kD nuclear trans-regulatory protein termed Tax that transcriptionally activates the HTLV-I long terminal repeat (LTR), as well as select [corrected] cellular and heterologous viral promoters. Tax does not bind DNA specifically but, rather, acts in a more indirect manner. Tax activation of the HTLV-I LTR is mediated through constitutively expressed cellular factors that bind to cAMP response elements (CREs) present within the 21-bp enhancers of the LTR. In contrast, Tax transactivation of the interleukin-2 receptor-alpha gene (IL-2R alpha) and LTR of the type 1 human immunodeficiency virus (HIV-1) involves the induced nuclear expression of NF-kappa B. We now report the identification of missense mutations within the tax gene that functionally segregate these two pathways of trans-activation. Additionally, we demonstrate that the carboxyl terminus of the Tax protein, despite its acidic and predicted alpha-helical structure, is completely dispensable for trans-activation through either of these transcription factor pathways. Finally, we demonstrate that mutations within a putative zinc finger domain disrupt the nuclear localization of Tax and abolish trans-activation. These results demonstrate that Tax trans-activation of viral and cellular promoters involves at least two mechanisms of host transcription factor activation and suggest that this activation is likely mediated through distinct functional domains.

Cellular transcription factors and regulation of IL-2 receptor gene expression by HTLV-I tax gene product.

Expression of the interleukin-2 receptor (IL-2R alpha) gene is activated by the transcriptional activator protein, Tax (previously referred to as the tat gene product), encoded by the human T-cell leukemia virus (HTLV-I). Multiple protein binding sites for specific DNA-protein interactions were identified over the upstream IL-2R alpha transcriptional regulatory sequences. However, only one region, which includes the sequence motif GGGGAATCTCCC, was required for activation by both the tax gene product and mitogenic stimulation. Remarkably, this sequence also bound the nuclear factor NF kappa B, which is important for induction of kappa-immunoglobulin gene expression. A model is presented whereby regulation of cellular gene expression by the HTLV-I tax gene product occurs via an indirect mechanism that may involve a post-translational modification of preexistent cellular transcription factors.