Tax-mediated NF-κB Activation Research Articles

The helicase-like transcription factor redirects the autophagic flux and restricts human T cell leukemia virus type 1 infection

Retroviruses and their host have coevolved in a delicate balance between viral replication and survival of the infected cell. In this equilibrium, restriction factors expressed by infected cells control different steps of retroviral replication such as entry, uncoating, nuclear import, expression, or budding. Here, we describe a mechanism of restriction against human T cell leukemia virus type 1 (HTLV-1) by the helicase-like transcription factor (HLTF). We show that RNA and protein levels of HLTF are reduced in primary T cells of HTLV-1-infected subjects, suggesting a clinical relevance. We further demonstrate that the viral oncogene Tax represses HLTF transcription via the Enhancer of zeste homolog 2 methyltransferase of the Polycomb repressive complex 2. The Tax protein also directly interacts with HLTF and induces its proteasomal degradation. RNA interference and gene transduction in HTLV-1-infected T cells derived from patients indicate that HLTF is a restriction factor. Restoring the normal levels of HLTF expression induces the dispersal of the Golgi apparatus and overproduction of secretory granules. By synergizing with Tax-mediated NF-κB activation, physiologically relevant levels of HLTF intensify the autophagic flux. Increased vesicular trafficking leads to an enlargement of the lysosomes and the production of large vacuoles containing viral particles. HLTF induction in HTLV-1-infected cells significantly increases the percentage of defective virions. In conclusion, HLTF-mediated activation of the autophagic flux blunts the infectious replication cycle of HTLV-1, revealing an original mode of viral restriction.

SQSTM1/p62 regulates HTLV-1 tax mediated NF-κB activation

HTLV-1-mediated cellular transformation relies on Tax-dependent activation of the NF-κB pathway, which has previously been shown to require Tax poly-ubiquitination and interaction with cellular factors, such as Optineurin (OPTN). The recent identification of OPTN as a selective autophagy receptor sharing high sequence similarities with sequestosome-1 (SQSTM-1/p62), a well-described selective autophagy receptor and NF-κB signaling adaptor, led us to hypothesize that Tax could hijack selective autophagy receptors for an efficient NF-κB activation. Using immunoprecipitation and confocal imaging of endogenous SQSTM/p62 in Tax-expressing cells or in HTLV-1 chronically infected T-cell lines, we showed that Tax interacts with SQSTM-1/p62. This interaction was independent of Tax ubiquitination and of the presence of the Tax PDZ-binding motif. Tax-mediated activation of NF-κB in p62-deficient cells was significantly reduced compared to wild type cells, indicating that SQSTM/p62 is necessary for Tax activity. Surprisingly however, over-expression of SQSTM-1/p62 or induction of autophagy led to a dramatic decrease in the amount of soluble Tax, along with a reduced induction of NF-κB. This suggests that Tax could be a substrate of selective autophagy. Altogether, our results reveal the double-edged consequences of Tax / SQSTM/p62 interaction, with the potentiation of Tax activity and the induction of Tax sequestration. They highlight the complex relationships that this viral protein has.

Human T Cell Leukemia Virus Type 2 Tax-Mediated NF-κB Activation Involves a Mechanism Independent of Tax Conjugation to Ubiquitin and SUMO

Permanent activation of the NF-κB pathway by the human T cell leukemia virus type 1 (HTLV-1) Tax (Tax1) viral transactivator is a key event in the process of HTLV-1-induced T lymphocyte immortalization and leukemogenesis. Although encoding a Tax transactivator (Tax2) that activates the canonical NF-κB pathway, HTLV-2 does not cause leukemia. These distinct pathological outcomes might be related, at least in part, to distinct NF-κB activation mechanisms. Tax1 has been shown to be both ubiquitinated and SUMOylated, and these two modifications were originally proposed to be required for Tax1-mediated NF-κB activation. Tax1 ubiquitination allows recruitment of the IKK-γ/NEMO regulatory subunit of the IKK complex together with Tax1 into centrosome/Golgi-associated cytoplasmic structures, followed by activation of the IKK complex and RelA/p65 nuclear translocation. Herein, we compared the ubiquitination, SUMOylation, and acetylation patterns of Tax2 and Tax1. We show that, in contrast to Tax1, Tax2 conjugation to endogenous ubiquitin and SUMO is barely detectable while both proteins are acetylated. Importantly, Tax2 is neither polyubiquitinated on lysine residues nor ubiquitinated on its N-terminal residue. Consistent with these observations, Tax2 conjugation to ubiquitin and Tax2-mediated NF-κB activation is not affected by overexpression of the E2 conjugating enzyme Ubc13. We further demonstrate that a nonubiquitinable, non-SUMOylable, and nonacetylable Tax2 mutant retains a significant ability to activate transcription from a NF-κB-dependent promoter after partial activation of the IKK complex and induction of RelA/p65 nuclear translocation. Finally, we also show that Tax2 does not interact with TRAF6, a protein that was shown to positively regulate Tax1-mediated activation of the NF-κB pathway.

NF-kappaB activation: Tax sumoylation is out, but what about Tax ubiquitination?

Activation of the NF-κB transcription factors by the viral protein Tax plays a vital role in the pathogenesis of diseases associated with human T-cell lymphotropic virus type I (HTLV-I). The Tax oncoprotein undergoes constitutive K63-linked ubiquitination and sumoylation; however, the roles and molecular mechanisms of these post-translational modifications in Tax-mediated NF-κB activation are being debated. Here, we discuss our current understanding of Tax activation of NF-κB, with a focus on the controversies and the challenges that we are facing.

An RNA Interference Screen Identifies the Deubiquitinase STAMBPL1 as a Critical Regulator of Human T-Cell Leukemia Virus Type 1 Tax Nuclear Export and NF-κB Activation

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein actively shuttles between the nucleus, where it interacts with transcriptional and splicing regulatory proteins, and the cytoplasm, where it activates NF-κB. Posttranslational modifications of Tax such as ubiquitination regulate its subcellular localization and hence its function; however, the regulation of Tax trafficking and NF-κB activation by host factors is poorly understood. By screening a deubiquitinating (DUB) enzyme small interfering RNA (siRNA) library, we identified the metalloprotease STAM-binding protein-like 1 (STAMBPL1) as a positive regulator of Tax-mediated NF-κB activation. Overexpression of wild-type STAMBPL1, but not a catalytically inactive mutant, enhanced Tax-mediated NF-κB activation, whereas silencing of STAMBPL1 with siRNA impaired Tax activation of both the canonical and noncanonical NF-κB signaling pathways. STAMBPL1 regulated Tax-induced NF-κB signaling indirectly by controlling Tax nuclear/cytoplasmic transport and was required for DNA damage-induced Tax nuclear export. Together, these results reveal that the deubiquitinase STAMBPL1 is a key regulator of Tax trafficking and function.

STAMBPL1 is a deubiquitinating enzyme that regulates HTLV-I Tax subcellular localization and NF-kB activation

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein is predominantly localized in the nuclei where it is associated with transcriptional and splicing regulatory proteins to form the Tax speckled structures (TSS). Tax is also distributed in the Golgi apparatus where it activates the IkB kinase (IKK) complex upstream of NF-κB, an essential transcription factor for Tax-induced immortalization of T lymphocytes. Lysine 63 (K63)-linked polyubiquitination of Tax is necessary for Tax to localize to the Golgi and activate NF-κB, whereas K48-linked polyubiquitination of Tax promotes its degradation in the nucleus. Therefore, post-translational modifications of Tax regulate its subcellular localization and hence its function, however the host factors that regulate Tax localization are unknown. Using a deubiquitinating (DUB) enzyme siRNA library we identified STAM-binding protein-like 1 (STAMBPL1) as a DUB that is required for Tax-mediated NF-κB activation. STAMBPL1 is a member of the JAMM (JAB1/MPN/MOV34 metalloenzyme) family of zinc metalloproteases, several of which have been previously implicated in the regulation of receptor endocytosis and trafficking. Overexpression of wild-type STAMBPL1, but not a catalytically inactive mutant, enhances Tax-mediated NF-κB activation and the induction of NF-κB target genes. Silencing of STAMBPL1 with siRNA inhibits Tax activation of both the canonical and noncanonical NF-κB signaling pathways. Furthermore, overexpression of STAMBPL1 enhances the stability of Tax by blocking its proteasomal degradation, whereas siRNA silencing of STAMBPL1 promotes Tax degradation. Confocal microscopy experiments reveal that STAMBPL1 controls the subcellular localization of Tax by promoting its nuclear export and subsequent trafficking to the Golgi where it activates IKK. Together these results suggest that the deubiquitinating enzyme STAMBPL1 regulates the localization and stability of Tax, and consequently the activation levels of NF-κB.

Association of HTLV Tax proteins with TAK1-binding protein 2 and RelA in calreticulin-containing cytoplasmic structures participates in Tax-mediated NF-κB activation

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.

Role of post-translational modifications of HTLV-1 Tax in NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered, is the etiological agent of adult-T-cell leukemia/lymphoma. The HTLV-1 encoded Tax protein is a potent oncoprotein that deregulates gene expression by constitutively activating nuclear factor-κB (NF-κB). Tax activation of NF-κB is critical for the immortalization and survival of HTLV-1-infected T cells. In this review, we summarize the present knowledge on mechanisms underlying Tax-mediated NF-κB activation, with an emphasis on post-translational modifications of Tax.

The Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Requires the Ubiquitin-Conjugating Enzyme Ubc13 for NF-κB Activation

Ubiquitination of the human T-cell leukemia virus 1 Tax oncoprotein provides an important regulatory mechanism that promotes the Tax-mediated activation of NF-kappaB. However, the type of polyubiquitin chain linkages and the host factors that are required for Tax ubiquitination have not been identified. Here, we demonstrate that Tax polyubiquitin chains are composed predominantly of lysine 63-linked chains. Furthermore, the ubiquitination of Tax is critically dependent on the E2 ubiquitin-conjugating enzyme Ubc13. Tax interacts with Ubc13, and small interfering RNA-mediated knockdown of Ubc13 expression abrogates Tax ubiquitination and the activation of NF-kappaB. Mouse fibroblasts lacking Ubc13 exhibit impaired Tax activation of NF-kappaB despite normal tumor necrosis factor- and interleukin-1-mediated NF-kappaB activation. Finally, the interaction of Tax with NEMO is disrupted in the absence of Tax ubiquitination and Ubc13 expression, suggesting that Tax ubiquitination is critical for NEMO binding. Collectively, our results reveal that Ubc13 is essential for Tax ubiquitination, its interaction with NEMO, and Tax-mediated NF-kappaB activation.

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

Activation of NF-κB by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of IκB Kinase

A Novel NF-κB Pathway Involving IKKβ and p65/RelA Ser-536 Phosphorylation Results in p53 Inhibition in the Absence of NF-κB Transcriptional Activity

Nuclear factor kappaB (NF-kappaB) plays an important role in regulating cellular transformation and apoptosis. The human T-cell lymphotropic virus type I protein, Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-kappaB. We have demonstrated previously that Tax inhibits p53 activity through the p65/RelA subunit of NF-kappaB. We now present evidence that suggests that the upstream kinase IKKbeta plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF) IKKbeta-/-cells did not support Tax-mediated p53 inhibition, whereas MEFs lacking IKKalpha allowed Tax inhibition of p53. Second, transfection of IKKbeta wild type (WT), but not a kinase-dead mutant, into IKKbeta-/-cells rescued p53 inhibition by Tax. Third, the IKKbeta-specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/-cells transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA Ser-536 phosphorylation in WT or IKKalpha-/- cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKKbeta-/-cells, suggesting a link between IKKbeta and p65/RelA phosphorylation. Consistent with this observation, blocking IKKbeta kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type I-transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-kappaB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-kappaB p65/RelA pathway that functions to inhibit p53 but does not require NF-kappaB transcription activity.

Human T cell leukemia virus type I Tax activates CD40 gene expression via the NF-kappa B pathway

The human T cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is etiologically linked to the genesis of adult T cell leukemia (ATL) as well as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Emerging evidence suggests that the pathogenicity of HTLV-I involves deregulated activation of immune cells, especially T lymphocytes, although the underlying mechanism remains unclear. In this study, we demonstrate that HTLV-I Tax induces the aberrant expression of CD40, a member of the tumor necrosis factor receptor (TNFR) family that plays an important role in lymphocyte activation and differentiation. In a panel of HTLV-I-transformed T cell lines analyzed, CD40 expression was highly elevated compared to HTLV-I-negative T cells. Using Tax mutants and a genetically manipulated T cell system, we demonstrated that Tax-induced CD40 expression required the NF-κB signaling pathway. In addition, ligation of CD40 on T cells with recombinant CD40L elicited NF-κB activation, suggesting that the CD40 pathway is intact and may participate in a positive regulatory loop in T cells. CD40 ligation strongly synergized with Tax to activate NF-κB, suggesting that CD40 signals may costimulate Tax-mediated NF-κB activation, particularly when Tax is expressed at low levels. Collectively, these results indicate that CD40 is a novel Tax-regulated gene, and the regulation of CD40 by Tax may play a role in cellular activation and HTLV-I-induced disease pathogenesis.

Human T-cell lymphotropic virus type 1 Tax represses c-Myb-dependent transcription through activation of the NF-kappaB pathway and modulation of coactivator usage.

The proto-oncogene c-myb is essential for a controlled balance between cell growth and differentiation. Aberrant c-Myb activity has been reported for numerous human cancers, and enforced c-Myb transcription can transform cells of lymphoid origin by stimulating cellular proliferation and inhibiting apoptotic pathways. Here we demonstrate that activation of the NF-kappaB pathway by the HTLV-1 Tax protein leads to transcriptional inactivation of c-Myb. This conclusion was supported by the fact that Tax mutants unable to stimulate the NF-kappaB pathway could not inhibit c-Myb transactivating functions. In addition, inhibition of Tax-mediated NF-kappaB activation by coexpression of IkappaBalpha restored c-Myb transcription, and Tax was unable to block c-Myb transcription in a NEMO knockout cell line. Importantly, physiological stimuli, such as signaling with the cellular cytokines tumor necrosis factor alpha, interleukin 1 beta (IL-1beta), and lipopolysaccharide, also inhibited c-Myb transcription. These results uncover a new link between extracellular signaling and c-Myb-dependent transcription. The mechanism underlying NF-kappaB-mediated repression was identified as sequestration of the coactivators CBP/p300 by RelA. Interestingly, an amino-terminal deletion form of p300 lacking the C/H1 and KIX domains and unable to bind RelA retained the ability to stimulate c-Myb transcription and prevented NF-kappaB-mediated repression.

Genome-wide expression changes induced by HTLV-1 Tax: evidence for MLK-3 mixed lineage kinase involvement in Tax-mediated NF-kappaB activation.

The Tax protein of human T-lymphotropic virus type 1 (HTLV-1), an oncoprotein that transactivates viral and cellular genes, plays a key role in HTLV-1 replication and pathogenesis. We used cDNA microarrays to examine Tax-mediated transcriptional changes in the human Jurkat T-cell lines JPX-9 and JPX-M which express Tax and Tax-mutant protein, respectively, under the control of an inducible promoter. Approximately 300 of the over 2000 genes examined were differentially expressed in the presence of Tax. These genes were grouped according to their function and are discussed in the context of existing findings in the literature. There was strong agreement between our results and genes previously reported as being Tax-responsive. Genes that were differentially expressed in the presence of Tax included those related to apoptosis, the cell cycle and DNA repair, signaling factors, immune modulators, cytokines and growth factors, and adhesion molecules. Functionally, we provide evidence that one of these genes, the mixed-lineage kinase MLK-3, is involved in Tax-mediated NF-kappa-B signaling. Our current results provide additional insights into Tax-mediated signaling.

NF-κB-inducing Kinase and IκB Kinase Participate in Human T-cell Leukemia Virus I Tax-mediated NF-κB Activation

The tax gene product of human T-cell leukemia virus I induces aberrant expression of various cellular genes, which contributes to transformation of host cells. Induction of many Tax target genes is mediated through transcription factor NF-kappaB. Here we show that Tax triggers activation of cellular protein kinases, IkappaB kinase alpha (IKKalpha) and IKKbeta, which phosphorylate the NF-kappaB inhibitory protein IkappaB alpha, resulting in its degradation and NF-kappaB activation. Constitutive IKK activation occurs in both Tax-transfected and human T-cell leukemia virus I-infected T cells. We further demonstrate that Tax-mediated NF-kappaB signaling also requires the NF-kappaB-inducing kinase (NIK). Consistently, inactive forms of either IKKs or NIK attenuate Tax-mediated NF-kappaB activation. Therefore, Tax activates NF-kappaB by targeting cellular signaling molecules, including both IKKs and NIK.