The transcription factor NF-B 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-B pathway by Tax involves its interaction with the regulatory subunit of the IB kinase (IKK) complex, NEMO/IKK␥, 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-B and free IKK␥ in these nuclear structures. However, this sumoylation step is not sufficient for the activation of the NF-B 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-B 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 IKK␥ in nuclear bodies for activation of gene expression. These modifications may result in targets for the treatment of adult T-cell leukemia.The human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia and a virus-associated neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (31,36,47). These two diseases have been linked to the expression of the HTLV-1 regulatory protein Tax (10,13,29,41). Tax is a potent transcriptional activator of viral genes as well as specific cellular genes and has a clear oncogenic potential since Tax is able to transform T lymphocytes and fibroblasts and induces tumors in transgenic mice (19). A substantial part of the oncogenic properties of Tax is associated with its ability to activate the expression of cellular genes that control T-cell proliferation and differentiation by inducing constitutive activation of the NF-B pathway (2, 38).In nonstimulated cells, inactive NF-B complexes composed of p50 and RelA heterodimers are retained in the cytoplasm by NF-B inhibitors of the IB family (15,23,40). Tax activation of the NF-B pathway involves its interaction with the regulatory subunit of the IB kinase (IKK) complex, NEMO/IKK␥, leading to the activation of the two catalytic subunits, IKK␣ and IKK␤ (8,14). Activation of the IB kinase complex by Tax determines the phosphorylation of the IB proteins, leading to their ubiquitination and degradation by the proteasome and to the migration of the NF-B complexes to the nucleus (18,45,46). It also determines the phosphorylation of the RelA subunit of NF-B, a prerequisite for activation of RelA and p50 heterodimers in the nucleus (30). However, Tax also colocalizes in discrete nuclear bodies (NB) with the two subunits of NF-B, p50 and RelA, in addition to RNA polymerase II, and the assembly of these nuclear structures correlates with Tax transcriptional activity (4-6, 37). Thus, Tax-mediated activation o...

Human T-cell leukemia virus type 1 (HTLV-1) is the retrovirus responsible for adult T-cell leukemia and HTLV-1-associated myelopathy. Adult T-cell leukemia development is mainly due to the ability of the viral oncoprotein Tax to promote T-cell proliferation, whereas the appearance of HTLV-1-associated myelopathy involves the antigenic properties of Tax. Understanding the events regulating the intracellular level of Tax is therefore an important issue. How Tax is degraded has not been determined, but it is known that Tax binds to proteasomes, the major sites for degradation of intracellular proteins, generally tagged through polyubiquitin conjugation. In this study, we investigated the relationship between Tax, ubiquitin, and proteasomes. We report that mono-and polyubiquitinated Tax proteins can be recovered from both transfected 293T cells and T lymphocytes. We also show that lysine residues located in the carboxy-terminal domain of Tax are the principal targets of this process. Remarkably, we further demonstrate that mutation of lysine residues in the C-terminal part of Tax, which massively reduces Tax ubiquitination, impairs proteasome binding, and conversely, that a Tax mutant that binds poorly to this particle (M22) is faintly ubiquitinated, suggesting that Tax ubiquitination is required for association with cellular proteasomes. Finally, we document that comparable amounts of ubiquitinated species were found whether proteasome activities were inhibited or not, providing evidence that they are not directly addressed to proteasomes for degradation. These findings indicate that although it is ubiquitinated and binds to proteasomes, Tax is not massively degraded via the ubiquitinproteasome pathway and therefore reveal that Tax conjugation to ubiquitin mediates a nonproteolytic function.Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia, a malignant monoclonal proliferation of CD4 ϩ T lymphocytes and of a chronic myelopathy called HTLV-1-associated myelopathy/tropical spastic paraparesis (36). Although these two diseases are definitely divergent in term of pathogenic mechanisms, the HTLV-1 Tax regulatory protein can be considered a key actor in both cases. First, via its ability to activate the viral promoter (31, 34), chronic Tax production is required to sustain viral replication. Second, HTLV-1-mediated immortalization of T lymphocytes, a fundamental event for subsequent cell transformation, results mainly from the ability of Tax to trigger T-cell proliferation through various mechanisms, including transcriptional transactivation of cellular genes (reviewed in reference 21) and promotion of cell cycle and deregulation of apoptosis (reviewed in reference 13).HTLV-1-associated myelopathy/tropical spastic paraparesis is not related to T-cell transformation and is considered as an immune-mediated pathology (reviewed in reference 15). Complex mechanisms are involved, among which exacerbation of the antiviral cytotoxic T-cell response (7, 23) and cross recognition of cell...

The oncogenic potential of the HTLV-1 Tax protein involves activation of the NF-κB pathway, which depends on Tax phosphorylation, ubiquitination and sumoylation. We demonstrate that the nuclei of Tax-expressing cells, including HTLV-1 transformed T-lymphocytes, contain a pool of Tax molecules acetylated on lysine residue at amino acid position 346 by the transcriptional coactivator p300. Phosphorylation of Tax on serine residues 300/301 was a prerequisite for Tax localization in the nucleus and correlated with its subsequent acetylation by p300, whereas sumoylation, resulting in the formation of Tax nuclear bodies in which p300 was recruited, favored Tax acetylation. Overexpression of p300 markedly increased Tax acetylation and the ability of a wild type HTLV-1 provirus, –but not of a mutant provirus carrying an acetylation deficient Tax gene–, to activate gene expression from an integrated NF-κB-controlled promoter. Thus, Tax acetylation favors NF-κB activation and might play an important role in HTLV-1-induced cell transformation.

HTLV-1 is more pathogenic than HTLV-2B. The difference is generally attributed to the properties of their individual transactivating Tax proteins. By using internal Flag-6His tagged Tax-1 and Tax-2B, which display transcriptional activities comparable to the untagged proteins and can be recognized by a single anti-Flag antibody, we demonstrate that Tax-2B is modified by ubiquitination and sumoylation. In addition, Tax2B is distributed in punctuate nuclear structures that include the RelA subunit of NF-kappaB, as has been previously demonstrated for Tax-1.

BackgroundRetroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway.ResultsThe comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1.ConclusionsBoth Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway.