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HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.

The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Retroviruses 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., Results: The 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., Conclusions: Both 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.

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. Since the viral transactivator Tax-1 is a major player in T-cell transformation, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and to counteract neoplastic transformation. We demonstrate that CIITA, the master regulator of major histocompatibility complex class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1 both when exogenously transfected in 293T cells and when endogenously expressed by a subset of U937 promonocytic cells. Tax-1 and CIITA physically interact in vivo via the first 108 amino acids of Tax-1 and two CIITA adjacent regions (amino acids 1 to 252 and 253 to 410). Interestingly, only CIITA 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64 to 124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2. Instead, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular coactivators p300/CBP-associated factor (PCAF), cyclic AMP-responsive element binding protein (CREB), and activating transcription factor 1 (ATF1), which are required for the optimal activation of HTLV-1 promoter. Accordingly, the overexpression of PCAF, CREB, and ATF1 restored Tax-1-dependent transactivation of the viral long-terminal-repeat promoter inhibited by CIITA. These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.

Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity.

HTLV-1 is more pathogenic than HTLV-2 despite having a similar genome and closely related transactivating oncoproteins. Both Tax-1 protein from HTLV-1 and Tax-2 from HTLV-2 activate the NF-κB pathway. The mechanisms involved in Tax-1 deregulation of this signalling pathway have been thoroughly investigated, but little is known about regulation by Tax-2. We have compared the interaction of Tax-1 and Tax-2 with two key NF-κB signalling factors: TAK1-binding protein 2 (TAB2), an adaptor involved in the activation of TAK1 kinase, and RelA, the active subunit of the canonical RelA/p50 NF-κB transcription factor. Tax-2 formed stable complexes with both RelA and TAB2. These two NF-κB factors colocalized with Tax proteins in dotted cytoplasmic structures targeted by calreticulin, a multi-process calcium-buffering chaperone. Co-expression of RelA and/or TAB2 markedly increased Tax-mediated NF-κB activation. These findings provide new insights into the role of RelA, TAB2 and Tax in the deregulation of the NF-κB pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The HTLV-1 transactivator Tax is an oncoprotein capable of deregulating the expression of many cellular genes and interfering with signalling pathways. Here we show that Tax-1 is phosphorylated in vitro by the pleiotropic human serine/threonine kinase CK2 at three residues, Ser-336, Ser-344 and Thr-351, close to and within its C-terminal PDZ-binding motif. We also show that the mutation of Thr-351 to aspartate abolishes Tax-1 binding to the scaffold protein hDlg, a tumour suppressor factor, while having no effect on transactivation. These results suggest that CK2, whose constitutive activity is often hijacked by viruses to sustain their vital cycle, could modulate Tax-1 oncogenic interactions.

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.

Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

The human T-cell leukemia virus type 2 (HTLV-2), an oncogenic retrovirus closely related to HTLV-1, produces a lifelong infection whose possible association to certain human diseases is still debated. Although some viral products can influence the expression and action of cellular genes, very little is known about the molecular mechanisms involved. Here we show that the AIR-1-encoded human major histocompatibility complex (MHC) class II transactivator (CIITA) strongly inhibits viral replication, but not virus entry, in human B- and T-cell susceptible targets. This effect results from CIITA inhibiting the Tax-mediated transactivation of the HTLV-2 long-term repeat. Further molecular analysis shows that the N-terminal region of CIITA encompassing the first 321 amino acids is responsible for the inhibitory effect on viral replication. This region is crucial for the transactivation of human MHC class II genes and includes the activation domain as well as domains interacting with coactivators that also are used by the viral transactivator Tax to modulate cellular functions. These results represent the first evidence that a cellular transcriptional activator, controlling the coordinate expression of the entire family of MHC class II antigen-presenting molecules, inhibits HTLV-2 viral replication by a distinct mechanism. In this new role CIITA may represent a new tool for therapeutic strategies aimed at counteracting HTLV-2 replication and spreading.