Peptide-MHC (pMHC) binding to a human antiviral T cell receptor induces long-range allosteric communication between pMHC- and CD3-binding sites.

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αß TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αß TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and ß chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cß FG loop projecting from the Vß/Cß interface; 2) a cluster of Cß residues near the Cß αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR ß chain enables allosteric communication between the TCR-binding sites for pMHC and CD3.

Simian T Lymphotropic Virus 1 Infection of Papio anubis: tax Sequence Heterogeneity and T Cell Recognition.

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons, animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus, our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons, extremely low heterogeneity of STLV sequences within each baboon, no evidence for superinfection within each baboon, and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed, premature stop codons were observed in 7% and 56% of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752, respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons, the identity of the viral gene product that is the major target of cellular immune responses, the persistence of viral amino acid sequences that are the major targets of cellular immune responses, and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts.

Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein.

Human T-cell leukemia virus (HTLV) type 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 domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.

Clinical outcomes of a novel therapeutic vaccine with Tax peptide-pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study.

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type-I (HTLV-I)-infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti-ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate- to high-risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax-specific CTL responses were observed with peaks at 16-20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide-pulsed DC vaccine is a safe and promising immunotherapy for ATL.

Conformational melding permits a conserved binding geometry in TCR recognition of foreign and self molecular mimics.

Molecular mimicry between foreign and self Ags is a mechanism of TCR cross-reactivity and is thought to contribute to the development of autoimmunity. The αß TCR A6 recognizes the foreign Ag Tax from the human T cell leukemia virus-1 when presented by the class I MHC HLA-A2. In a possible link with the autoimmune disease human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis, A6 also recognizes a self peptide from the neuronal protein HuD in the context of HLA-A2. We found in our study that the complexes of the HuD and Tax epitopes with HLA-A2 are close but imperfect structural mimics and that in contrast with other recent structures of TCRs with self Ags, A6 engages the HuD Ag with the same traditional binding mode used to engage Tax. Although peptide and MHC conformational changes are needed for recognition of HuD but not Tax and the difference of a single hydroxyl triggers an altered TCR loop conformation, TCR affinity toward HuD is still within the range believed to result in negative selection. Probing further, we found that the HuD-HLA-A2 complex is only weakly stable. Overall, these findings help clarify how molecular mimicry can drive self/nonself cross-reactivity and illustrate how low peptide-MHC stability can permit the survival of T cells expressing self-reactive TCRs that nonetheless bind with a traditional binding mode.

Dimerization and a novel Tax speckled structure localization signal are required for Tax nuclear localization.

The human T-cell leukemia virus type 1 oncoprotein Tax has pleiotropic activities, a subset of which likely leads to immortalization of T cells. Tax is expressed and known to function in both the cell nucleus and the cytoplasm. Tax has defined nuclear localization (NLS) and nuclear export signals that enable shuttling between the two compartments. In this study, we identified a novel region in Tax that targets the protein to discrete nuclear foci that we have previously termed Tax speckled structures (TSS). We demonstrated that the identified region is both necessary and sufficient for directing proteins to TSS. This novel TSS localization signal (TSLS), spanning amino acids 50 to 75, is separable from and adjacent to the NLS of Tax. Coexpression of a Tax NLS mutant and a Tax TSLS mutant rescued the nuclear entry and subnuclear TSS targeting of both proteins, demonstrating that these signals are independent domains. Our analysis also revealed that Tax proteins deficient for dimerization fail to localize to the nucleus. Consequently, when we restored dimerization via induction of a heterologous "dimerizer" domain, nuclear localization was rescued. Thus, we defined additional domains in Tax specific for nuclear localization and subnuclear targeting. Our results reveal a more complex network for regulation of Tax subcellular localization and subsequent function.

Fluorine substitutions in an antigenic peptide selectively modulate T-cell receptor binding in a minimally perturbing manner.

TCR (T-cell receptor) recognition of antigenic peptides bound and presented by MHC (major histocompatibility complex) molecules forms the basis of the cellular immune response to pathogens and cancer. TCRs bind peptide-MHC complexes weakly and with fast kinetics, features which have hindered detailed biophysical studies of these interactions. Modified peptides resulting in enhanced TCR binding could help overcome these challenges. Furthermore, there is considerable interest in using modified peptides with enhanced TCR binding as the basis for clinical vaccines. In the present study, we examined how fluorine substitutions in an antigenic peptide can selectively impact TCR recognition. Using a structure-guided design approach, we found that fluorination of the Tax peptide [HTLV (human T-cell lymphotropic virus)-1 Tax(11-19)] enhanced binding by the Tax-specific TCR A6, yet weakened binding by the Tax-specific TCR B7. The changes in affinity were consistent with crystallographic structures and fluorine chemistry, and with the A6 TCR independent of other substitutions in the interface. Peptide fluorination thus provides a means to selectively modulate TCR binding affinity without significantly perturbing peptide composition or structure. Lastly, we probed the mechanism of fluorine's effect on TCR binding and we conclude that our results were most consistent with a 'polar hydrophobicity' mechanism, rather than a purely hydrophobic- or electrostatic-based mechanism. This finding should have an impact on other attempts to alter molecular recognition with fluorine.

A general and efficient approach for NMR studies of peptide dynamics in class I MHC peptide binding grooves.

T-Cell receptor recognition of peptides bound by major histocompatibility complex (MHC) proteins initiates a cellular immune response. Dynamics of peptides within MHC binding grooves can influence TCR recognition, yet NMR studies which could address this rigorously have been hindered by the expense of isotopically labeled peptides and the large size of peptide-MHC complexes. Here we describe a methodology for characterizing peptide dynamics within MHC binding grooves via NMR, using a biosynthetic approach for producing labeled peptide. With the Tax(11-19) peptide bound to the human class I MHC HLA-A*0201, we demonstrate that peptide generated in this manner can be well characterized in MHC binding grooves by NMR, providing opportunities to more precisely study the role of peptide dynamics in TCR recognition. Demonstrating the utility of such studies, the data with the Tax(11-19) peptide indicate the presence of slow conformational exchange in the peptide, supporting an "induced-fit" style TCR binding mechanism.

HTLV-1 Tax-1 interacts with SNX27 to regulate cellular localization of the HTLV-1 receptor molecule, GLUT1.

An estimated 10-20 million people worldwide are infected with human T cell leukemia virus type 1 (HTLV-1), with endemic areas of infection in Japan, Australia, the Caribbean, and Africa. HTLV-1 is the causative agent of adult T cell leukemia (ATL) and HTLV-1 associated myopathy/tropic spastic paraparesis (HAM/TSP). HTLV-1 expresses several regulatory and accessory genes that function at different stages of the virus life cycle. The regulatory gene Tax-1 is required for efficient virus replication, as it drives transcription of viral gene products, and has also been demonstrated to play a key role in the pathogenesis of the virus. Several studies have identified a PDZ binding motif (PBM) at the carboxyl terminus of Tax-1 and demonstrated the importance of this domain for HTLV-1 induced cellular transformation. Using a mass spectrometry-based proteomics approach we identified sorting nexin 27 (SNX27) as a novel interacting partner of Tax-1. Further, we demonstrated that their interaction is mediated by the Tax-1 PBM and SNX27 PDZ domains. SNX27 has been shown to promote the plasma membrane localization of glucose transport 1 (GLUT1), one of the receptor molecules of the HTLV-1 virus, and the receptor molecule required for HTLV-1 fusion and entry. We postulated that Tax-1 alters GLUT1 localization via its interaction with SNX27. We demonstrate that over expression of Tax-1 in cells causes a reduction of GLUT1 on the plasma membrane. Furthermore, we show that knockdown of SNX27 results in increased virion release and decreased HTLV-1 infectivity. Collectively, we demonstrate the first known mechanism by which HTLV-1 regulates a receptor molecule post-infection.

The HTLV-1 Tax interactome.

The Tax1 oncoprotein encoded by Human T-lymphotropic virus type I is a major determinant of viral persistence and pathogenesis. Tax1 affects a wide variety of cellular signalling pathways leading to transcriptional activation, proliferation and ultimately transformation. To carry out these functions, Tax1 interacts with and modulates activity of a number of cellular proteins. In this review, we summarize the present knowledge of the Tax1 interactome and propose a rationale for the broad range of cellular proteins identified so far.

Molecular characterization of HTLV-1 Tax interaction with the KIX domain of CBP/p300.

The viral oncoprotein Tax mediates transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1). Both Tax and the cellular transcription factor CREB bind to viral cyclic AMP response elements (vCREs) located in the viral promoter. Tax and serine 133 phosphorylated CREB (pCREB) bound to the HTLV-1 promoter facilitate viral transcription via the recruitment of the large cellular coactivators CBP/p300. While the interaction between the phosphorylated kinase inducible domain (pKID) of pCREB and the KIX domain of CBP/p300 has been well characterized, the molecular interactions between KIX, full-length Tax, and pCREB have not been examined. Here we biochemically characterized the interaction between Tax and KIX in a physiologically relevant complex containing pCREB and vCRE DNA. Our data show that Tax and pCREB simultaneously and independently bind two distinct surfaces on the KIX domain: Tax binds KIX at the previously characterized mixed-lineage leukemia (MLL) protein interaction surface while pCREB binds KIX at the pKID-KIX interface. These results provide evidence for a model in which Tax and pCREB bind distinct surfaces of KIX for effective CBP/p300 recruitment to the HTLV-1 promoter. We also show that MLL competes with Tax for KIX binding, suggesting a novel mechanism of Tax oncogenesis in which normal MLL function is disrupted by Tax.

The tax protein from the primate T-cell lymphotropic virus type 3 is expressed in vivo and is functionally related to HTLV-1 Tax rather than HTLV-2 Tax.

Human T-cell leukemia virus and simian T-cell leukemia virus (STLV) form the primate T-cell lymphotropic viruses group. Human T-cell leukemia virus type 1 and type 2 (HTLV-1 and HTLV-2) encode the Tax viral transactivator (Tax1 and Tax2, respectively). Tax1 possesses an oncogenic potential and is responsible for cell transformation both in vivo and in vitro. We and others have recently discovered the existence of human T-cell lymphotropic virus type 3. However, there is currently no evidence for the presence of a Tax protein in HTLV-3-infected individuals. We show that the serum of an HTLV-3 asymptomatic carrier and the sera of two STLV-3-infected monkeys contain specific anti-Tax3 antibodies. We also show that tax3 mRNA is present in the PBMCs obtained from an STLV-3-infected monkey, demonstrating that Tax3 is expressed in vivo. We further demonstrate that Tax3 intracellular localization is very similar to that of Tax1 and that Tax3 binds to both CBP and p300 coactivators. Using purified Tax3, we show that the protein increases transcription from a 4TxRE G-free cassette plasmid in an in vitro transcription assay. In all cell types tested, including transiently transfected lymphocytes, Tax3 activates its own promoter STLV-3 long terminal repeat (LTR), which contains only two Tax Responsive Elements (TREs), and activates also HTLV-1 and HTLV-2 LTRs. In addition, Tax3 also activates the NF-kappaB pathway. We also show that Tax3 possesses a PDZ-binding sequence at its C-terminal end. Our results demonstrate that Tax3 is a transactivator, and that its properties are more similar to that of Tax1, rather than of Tax2. This suggests the possible occurrence of lymphoproliferative disorders among HTLV-3-infected populations.

Identification of a common docking topology with substantial variation among different TCR-peptide-MHC complexes.

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.

T cell receptor recognition via cooperative conformational plasticity.

Although T cell receptor cross-reactivity is a fundamental property of the immune system and is implicated in numerous autoimmune pathologies, the molecular mechanisms by which T cell receptors can recognize and respond to diverse ligands are incompletely understood. In the current study we examined the response of the human T cell lymphotropic virus-1 (HTLV-1) Tax-specific T cell receptor (TCR) A6 to a panel of structurally distinct haptens coupled to the Tax 11-19 peptide with a lysine substitution at position 5 (Tax5K, LLFG[K-hapten]PVYV). The A6 TCR could cross-reactively recognize one of these haptenated peptides, Tax-5K-4-(3-Indolyl)-butyric acid (IBA), presented by HLA-A*0201. The crystal structures of Tax5K-IBA/HLA-A2 free and in complex with A6 reveal that binding is mediated by a mechanism of cooperative conformational plasticity involving conformational changes on both sides of the protein-protein interface, including the TCR complementarity determining region (CDR) loops, Valpha/Vbeta domain orientation, and the hapten-modified peptide. Our findings illustrate the complex role that protein dynamics can play in TCR cross-reactivity and highlight that T cell receptor recognition of ligand can be achieved through diverse and complex molecular mechanisms that can occur simultaneously in the interface, not limited to molecular mimicry and CDR loop shifts.

HTLV-1a tax gene and long terminal repeat sequences from Argentinean strains reveal disagreement with tax restriction fragment length polymorphism subtyping.

Sequence and cluster analysis have shown two HTLV-1a tax gene subgroups, tax A and tax B, which are related to long terminal repeat (LTR) molecular subtypes. On the basis of subgroup-specific nucleotide substitutions, restriction fragment length polymorphism (RFLP) analysis of the tax gene for subtyping HTLV-1a isolates was proposed. In this study we genetically characterized the tax gene from 63 HTLV-1-positive Argentinean individuals, including 14 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis and 49 healthy HTLV-1 carriers. RFLP analysis showed that 48 samples yielded the tax A profile (76.19%) and that 15 samples contained the uncut tax B profile (23.81%). However, the LTR and tax sequence analysis revealed that in fact only 2 from the 15 samples belonged to the HTLV-1aB subgroup, presenting four tax B subgroup-specific nucleotide substitutions. The tax gene cluster analysis also confirmed that the majority of Argentinean strains belonged to the Transcontinental HTLV-1aA subgroup. These results indicate that the tax gene RFLP assay which has been proposed and used by some authors to screen HTLV-1a subgroups, is not a suitable tool to perform molecular epidemiological characterization of HTLV-1a populations.

Complex formation between CREB and Tax enhances the binding affinity of CREB for the human T-cell leukemia virus type 1 21-base-pair repeats.

The regulation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is dependent on three cis-acting elements, known as the 21-bp repeats, in the long terminal repeat. Each of the 21-bp repeats contains a nonpalindromic cyclic AMP response element (CRE) sequence which is capable of binding members of the ATF/CREB family of transcription factors. The HTLV-1 transactivator protein Tax is able to markedly stimulate the in vitro binding of CREB to the CRE sites present in each of the 21-bp repeats but not to CRE sites present in cellular promoters. The ability to Tax to stimulate CREB binding to different CRE sites correlates with the ability of Tax to activate gene expression from these sites. We wished to determine how sequence differences between the somatostatin CRE and the 21-bp repeat were involved in this different response to Tax. Scatchard analysis indicated that CREB bound to the somatostatin CRE with a single class of high-affinity binding while CREB bound to the 21-bp repeats with a biphasic binding pattern, indicating the presence of both low- and high-affinity binding. Tax increased the affinity of CREB binding but not that of another ATF/CREB protein, CREB2, to the 21-bp repeat. However, Tax did not increase affinity of binding of CREB to the somatostatin CRE. To determine the mechanism by which Tax increased dCREB binding affinity, immobilized oligonucleotides corresponding to either the 21-bp repeat or the somatostatin CRE were used to demonstrate that Tax formed a highly specific complex with CREB on the 21-bp repeat but not on the somatostatin CRE. These results indicate that formation of a complex between Tax and CREB results in specific high-affinity binding of this ternary complex to the HTLV-1 21 bp repeats.

An exposed KID-like domain in human T-cell lymphotropic virus type 1 Tax is responsible for the recruitment of coactivators CBP/p300.

Human T-cell lymphotropic virus type 1 (HTLV-1) transcriptional activation is mediated by the viral transactivator, Tax, and three 21-bp repeats (Tax response element [TxRE]) located in the U3 region of the viral long terminal repeat (LTR). Each TxRE contains a core cyclic AMP response element (CRE) flanked by 5' G-rich and 3' C-rich sequences. The TxRE binds CREB (CRE-binding protein) and Tax to form a ternary complex and confers Tax-dependent transactivation. Recent data indicate that Tax functions as a specific link to connect CREB-binding protein (CBP)/p300 in a phosphorylation-independent manner to CREB/ATF-1 assembled on the viral 21-bp repeats. Glutathione S-transferase pull-down performed with Tax deletion mutants and peptide competition have localized the site in Tax critical for binding CBP/p300 to a highly protease-sensitive region around amino acid residues 81 to 95 (81QRTSKTLKVLTPPIT95) which lies between the domains previously proposed to be important for CREB binding and Tax subunit dimerization. Amino acid residues around the trypsin- and chymotrypsin-sensitive sites (88KVL90) of Tax bear resemblance to those in the kinase-inducible domain of CREB (129SRRPSYRKILNE140) surrounding Ser-133, which undergoes signal-induced phosphorylation to recruit CBP/p300. Site-directed mutagenesis of residues in this domain (R82A, K85A, K88A, and V89A) resulted in proteins which failed to transactivate from the HTLV-1 LTR in vivo. These mutants (K85A, K88A, and V89A) bind CREB with similar affinities as wild-type Tax, yet interaction with CBP/p300 is abrogated in various biochemical assays, indicating that the recruitment of CBP/p300 is crucial for Tax transactivation. A Tax mutant, M47, defective in the COOH-terminal transactivation domain, continued to interact with CBP/p300, suggesting that interactions with additional cellular factors are required for proper Tax function.

Functional activities of the human T-cell leukemia virus type I Tax oncoprotein: cellular signaling through NF-kappa B.

Human T-cell leukemia virus type I (HTLV-I) is the etiological agent for adult T-cell leukemia (ATL), as well as for tropical spastic paraparesis (TSP) and HTLV-I associate myelopathy (HAM). A biological understanding of the involvement of HTLV-I and in ATL has focused significantly on the workings of the virally-encoded 40 kDa phospho-oncoprotein, Tax. Tax is a transcriptional activator. Its ability to modulate the expression and function of many cellular genes has been reasoned to be a major contributory mechanism explaining HTLV-I-mediated transformation of cells. In activating cellular gene expression, Tax impinges upon several cellular signal-transduction pathways, including those for CREB/ATF and NF-kappa B. In this paper, we review aspects of Tax's transcriptional potential with particular focus on recent evidence linking Tax to IKK (I kappa B-kinase)-complex and MAP3Ks (mitogen-activated protein kinase kinase kinases).

The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation.

Human T-cell leukemia virus type-I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL), is estimated to affect 10-20 million people worldwide. The transforming ability of HTLV-I has been largely attributed to the viral protein Tax, which modulates the activity of several well-known cell cycle regulators. An important cell cycle regulator, the retinoblastoma (Rb) protein, is often inactivated in many cancers including virally induced cancers. Upon examination of Rb status, we observed a decrease in Rb protein expression in HTLV-1-infected cell lines as well as in ex vivo ATL patient samples. Transient transfection assays indicated that decreased Rb protein levels were Tax dependent. Here, we demonstrate for the first time that Tax directly associates with Rb. This interaction was localized within the B pocket of Rb and the C-terminus of Tax (aa 245-353). Within the C-terminus of Tax, we have identified an LXCXE-like motif, that when mutated resulted in the loss of Tax/Rb interaction. Furthermore, through the use of proteasome inhibitors, such as MG-132, in vivo and proteasome degradation assays in vitro, we found that Tax destabilizes the hypo-phosphorylated (active) form of Rb via the proteasome pathway. Therefore, we propose a model whereby Tax targets Rb to the proteasome by acting as a molecular bridge bringing Rb into contact with the proteasome for degradation.