Tax Oncoprotein Research Articles

Therapeutic advances for the management of adult T cell leukemia: Where do we stand?

Adult T cell leukemia (ATL) is an aggressive blood malignancy secondary to chronic infection with the human T cell leukemia virus type I (HTLV-1) retrovirus. ATL encompasses four subtypes (acute, lymphoma, chronic, and smoldering), which exhibit different clinical characteristics and respond differently to various treatment strategies. Yet, all four subtypes are characterized by a dismal long-term prognosis and a low survival rate. While antiretroviral therapy improves overall survival outcomes in smoldering and chronic subtypes, survival remains poor in lymphoma subtypes despite their good response to intensive chemotherapy. Nonetheless, acute ATL remains the most aggressive form associated with profound immunosuppression, chemo-resistance and dismal prognosis. Targeted therapies such as monoclonal antibodies, epigenetic therapies, and arsenic/IFN, emerged as promising therapeutic approaches in ATL. Allogeneic hematopoietic cell transplantation is the only potentially curative modality, alas applicable to only a small percentage of patients. The recent findings demonstrating the expression of the viral oncoprotein Tax in primary ATL cells from patients with acute or chronic ATL, albeit at low levels, and their dependence on continuous Tax expression for their survival, position ATL as a virus-addicted leukemia and validates the rationale of anti-viral treatment strategies. This review provides a comprehensive overview on conventional, anti-viral and targeted therapies of ATL, with emphasis on Tax-targeted therapied in the pre-clinical and clinical settings.

The tyrosine kinase KDR is essential for the survival of HTLV-1-infected T cells by stabilizing the Tax oncoprotein

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia/lymphoma (ATLL) and the neuroinflammatory disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax oncoprotein regulates viral gene expression and persistently activates NF-κB to maintain the viability of HTLV-1-infected T cells. Here, we utilize a kinome-wide shRNA screen to identify the tyrosine kinase KDR as an essential survival factor of HTLV-1-transformed cells. Inhibition of KDR specifically induces apoptosis of Tax expressing HTLV-1-transformed cell lines and CD4 + T cells from HAM/TSP patients. Furthermore, inhibition of KDR triggers the autophagic degradation of Tax resulting in impaired NF-κB activation and diminished viral transmission in co-culture assays. Tax induces the expression of KDR, forms a complex with KDR, and is phosphorylated by KDR. These findings suggest that Tax stability is dependent on KDR activity which could be exploited as a strategy to target Tax in HTLV-1-associated diseases.

PCBP1 interacts with the HTLV-1 Tax oncoprotein to potentiate NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma. The HTLV-1 Tax constitutively activates nuclear factor-κB (NF-κB) to promote the survival and transformation of HTLV-1-infected T cells. Despite extensive study of Tax, how Tax interacts with host factors to regulate NF-κB activation and HTLV-1-driven cell proliferation is not entirely clear. Here, we showed that overexpression of Poly (rC)-binding protein 1 (PCBP1) promoted Tax-mediated IκB kinase (IKK)-NF-κB signaling activation, whereas knockdown of PCBP1 attenuated Tax-dependent IKK-NF-κB activation. However, Tax activation of HTLV-1 long terminal repeat was unaffected by PCBP1. Furthermore, depletion of PCBP1 led to apoptosis and reduced proliferation of HTLV-1-transformed cells. Mechanistically, PCBP1 interacted and co-localized with Tax in the cytoplasm, and PCBP1 KH3 domain was indispensable for the interaction between PCBP1 and Tax. Moreover, PCBP1 facilitated the assembly of Tax/IKK complex. Collectively, our results demonstrated that PCBP1 may exert an essential effect in Tax/IKK complex combination and subsequent NF-κB activation, which provides a novel insight into the pathogenetic mechanisms of HTLV-1.

Gene-to-gene coordinated regulation of transcription and alternative splicing by 3D chromatin remodeling upon NF-κB activation.

The NF-κB protein p65/RelA plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA regulates gene transcription and alternative splicing through promoter enrichment and genomic exon occupancy, respectively. The intricate ways in which p65/RelA simultaneously governs these functions across various genes remain to be fully elucidated. In this study, we employed the HTLV-1 Tax oncoprotein, a potent activator of NF-κB, to investigate its influence on the three-dimensional organization of the genome, a key factor in gene regulation. We discovered that Tax restructures the 3D genomic landscape, bringing together genes based on their regulation and splicing patterns. Notably, we found that the Tax-induced gene-gene contact between the two master genes NFKBIA and RELA is associated with their respective changes in gene expression and alternative splicing. Through dCas9-mediated approaches, we demonstrated that NFKBIA-RELA interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on RELA. Our findings shed light on new regulatory mechanisms upon HTLV-1 Tax and underscore the integral role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and splicing levels in the context of the 3D genome.

Primary cells from patients with adult T cell leukemia/lymphoma depend on HTLV-1 Tax expression for NF-κB activation and survival

Adult T cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic infection with human T cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax initiates T cell transformation through activation of critical cellular pathways, including NF-κB. Unexpectedly, Tax protein is not detectable in most ATL cells, in contrast to the HTLV-1 HBZ protein which antagonizes Tax effects. Here, we demonstrate that primary ATL cells from patients with acute or chronic ATL express very low levels of Tax mRNA and protein. Critically, survival of these primary ATL cells is dependent on continued Tax expression. Mechanistically, Tax extinction results in reversal of NF-κB activation, P53/PML activation and apoptosis. Tax drives interleukin-10 (IL-10) expression and recombinant IL-10 rescues the survival of tax-depleted primary ATL cells. These results demonstrate the critical role of continued Tax and IL-10 expression for the survival of primary ATL cells, highlighting their relevance as therapeutic targets.

Alternative NF-κB Signaling Discriminates Induction of the Tumor Marker Fascin by the Viral Oncoproteins Tax-1 and Tax-2 of Human T-Cell Leukemia Viruses.

Simple SummaryThe actin-bundling protein Fascin is upregulated in many types of cancers, including adult T-cell leukemia/lymphoma, a tumor induced by the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1). Transcriptional regulation of Fascin is heterogeneous between different cell types and tissues, and Fascin is usually expressed at low levels in T-cells. We have previously shown that a single viral oncoprotein, Tax-1 of HTLV-1, is a potent inducer of Fascin in T-cells depending on classical NF-κB signaling. In this study, we discovered that transcriptional activation of Fascin by viral oncoproteins depends on activity of both the classical and the alternative NF-κB signaling cascade. Comparisons between Tax-1 and Tax-2 from the closely related but non-oncogenic HTLV-2 revealed that alternative NF-κB signaling discriminates transcriptional induction of Fascin by the Tax proteins encoded by HTLVs. Together, Tax-1 and Tax-2 proteins are useful tools to study oncogenic signaling in T-cells.Transcriptional regulation of the actin-bundling protein and tumor marker Fascin is highly diverse depending on cell and tumor type. Previously, we discovered that the viral oncoprotein Tax-1 of human T-cell leukemia virus type 1 (HTLV-1) considerably enhances Fascin expression in T-cells, depending on classical NF-κB signaling. In this study, we asked if the non-oncogenic Tax-2 of the related HTLV-2 is still able to induce Fascin by using luciferase assays, immunoblot, and qPCR. We found that Tax-2 only slightly induces Fascin expression compared to Tax-1; however, both Tax-1 and Tax-2 comparably activated a 1.6 kb fragment in the human Fascin promoter including Tax-responsive elements. Furthermore, we identified a link between Tax-induced activity of the alternative NF-κB pathway and Fascin induction. While treatment with the second mitochondria-derived activator of caspases (SMAC)-mimetic AZD5582, a compound known to robustly activate alternative NF-κB signaling, did not induce Fascin, combination of AZD5582 with activation of classical NF-κB signaling by Tax-2 significantly induced Fascin expression. In conclusion, our data demonstrate that both classical and alternative NF-κB activity are necessary for strong Fascin induction by the viral Tax oncoproteins, thus, shedding new light on the regulation of Fascin in T-cells and during viral transformation.

Human T-cell lymphotropic virus type 1 oncogenesis and cell-to-cell spread

Exploring host-HTLV-1 interactions and the molecular processes underpinning HTLV-1-mediated carcinogenesis is crucial for establishing effective treatments for viral infection and associated leukemia/lymphoma. Several HTLV-1 proteins have been shown to play important roles in the cellular transformation and immortalization of infected T cells. Through interactions with MAVS, STING, and RIP1, the HTLV-1 oncoprotein Tax suppresses the innate IFN response, resulting in the inhibition of TBK1-mediated phosphorylation of IRF3/IRF7. The HTLV-1 protein HBZ affects genomic integrity and inhibits target cell death and autophagy. Furthermore, it has been discovered that HBZ promotes the growth of ATL cells and aids in the evasion of infected cells from immunosurveillance. It currently appears that the efficacy of an individual's cytotoxic T cell (CTL) response to HTLV-1 is the most important single predictor of that person's provirus load, which can differ by more than 10,000-fold amongst HTLV-1-infected persons. We examine recent improvements in our knowledge of the pathophysiology, or underlying processes, of the illness produced by HTLV-1 infection in this article. Furthermore, we explore the future approach for targeting HTLV-1-associated malignancies and anti-HTLV-1 therapies. The pathogenic agent of adult T-cell leukemia/lymphoma (ATL) is human T-cell lymphotropic virus type 1 (HTLV-1). ATL is a fast-developing clonal tumour of CD4+ T cells which are cellular and viral protein interactions and pave the way for the discovery of new classes of cellular modulators, which may induce Tax oncogenesis and its impact on survival signalling pathways such as the NF-B and PI3K-Akt pathways, therapeutic target opportunities for ATL have been presented in two collaborative studies.

Epigenetic Regulation of Human T-Cell Leukemia Virus Gene Expression.

Viral and cellular gene expression are regulated by epigenetic alterations, including DNA methylation, histone modifications, nucleosome positioning, and chromatin looping. Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus associated with inflammatory disorders and T-cell lymphoproliferative malignancy. The transforming activity of HTLV-1 is driven by the viral oncoprotein Tax, which acts as a transcriptional activator of the cAMP response element-binding protein (CREB) and nuclear factor kappa B (NFκB) pathways. The epigenetic effects of Tax and the induction of lymphoproliferative malignancy include alterations in DNA methylation and histone modifications. In addition, alterations in nucleosome positioning and DNA looping also occur in HTLV-1-induced malignant cells. A mechanistic definition of these effects will pave the way to new therapies for HTLV-1-associated disorders.

ORP4L is a prerequisite for the induction of T-cell leukemogenesis associated with human T-cell leukemia virus 1

AbstractHuman T-cell leukemia virus 1 (HTLV-1) causes adult T-cell leukemia (ATL), but the mechanism underlying its initiation remains elusive. In this study, ORP4L was expressed in ATL cells but not in normal T-cells. ORP4L ablation completely blocked T-cell leukemogenesis induced by the HTLV-1 oncoprotein Tax in mice, whereas engineering ORP4L expression in T-cells resulted in T-cell leukemia in mice, suggesting the oncogenic properties and prerequisite of ORP4L promote the initiation of T-cell leukemogenesis. For molecular insight, we found that loss of miR-31 caused by HTLV-1 induced ORP4L expression in T-cells. ORP4L interacts with PI3Kδ to promote PI(3,4,5)P3 generation, contributing to AKT hyperactivation; NF-κB–dependent, p53 inactivation-induced pro-oncogene expression; and T-cell leukemogenesis. Consistently, ORP4L ablation eliminates human ATL cells in patient-derived xenograft ATL models. These results reveal a plausible mechanism of T-cell deterioration by HTLV-1 that can be therapeutically targeted.

FX1, a BCL6 inhibitor, reactivates BCL6 target genes and suppresses HTLV-1-infected T cells.

Human T cell leukemia virus type 1 (HTLV-1) is responsible for adult T cell leukemia (ATL); however, molecular and cellular mechanisms underlying HTLV-1-induced leukemogenesis are unclear. BCL6 oncogene is involved in cancer progression and a preferred target of anti-cancer treatments. Here, we aimed to evaluate BCL6 expression and the effects of BCL6 inhibitor (FX1) on HTLV-1-infected T cell lines. BCL6 expression was higher in HTLV-1-infected T cell lines than that in uninfected T cell lines. BCL6 was localized mostly in the nucleus. The virus oncoprotein Tax induced BCL6 mRNA expression in T cells, whereas BCL6 knockdown reduced HTLV-1-infected T cell proliferation; thus, confirmed the association of BCL6 with cancer progression. Further, FX1 efficiently inhibited the cell growth and survival of HTLV-1-infected T cell lines in a dose- and time-dependent manner. The decreased levels of cell cycle regulatory proteins (phosphorylated retinoblastoma protein, cyclin-dependent kinase 4, cyclin D2 and c-Myc) and the increased levels of BCL6 target proteins (p21, p27 and p53) showed that FX1 arrested cell cycle at the G1 phase. Apoptosis was induced concomitantly with Bak upregulation and downregulation of survivin, Bcl-xL and Mcl-1, as well as with the activation of caspase-3, -8, -9 and poly(ADP-ribose) polymerase. FX1 also inhibited NF-κB and Akt signaling pathways. These events were because of the induction of the activity of cell cycle checkpoint proteins and relief of direct repression of the targets of cell cycle checkpoint proteins. Thus, BCL6 might be considered a novel target for ATL treatment.

Arsenic Trioxide and Interferon-Alpha Eradicate Adult T Cell Leukemia Initiating Cells through PML/p53 Senescence and Activation of Innate Immunity

Adult T cell leukemia (ATL) is an aggressive chemo-resistant T cell malignancy secondary to chronic HTLV-I infection. The viral oncoprotein Tax initiates ATL in transgenic mice. We previously showed that arsenic trioxide (As) and interferon-α (IFN) combination, known to trigger proteasome mediated Tax degradation and apoptosis in ATL cells, cures murine ATL through clearance of leukemia initiating cells (LIC). We also showed that the triple combination of As, IFN and zidovudine is highly effective in the treatment of chronic ATL patients, inducing a shift from an immunosuppressive Treg/Th2 profile at diagnosis (high IL-10; low IL2; low IFN-γ) towards a Th-1 immunocompetent profile (high IFN-γ; high IL2; low IL-10).To dissect the molecular basis of As/IFN-induced ATL LIC eradication, primary ATL SCID mice were treated with As/IFN for 3 days. Spleen derived ATL cells were injected into secondary recipient SCID mice left untreated and sacrificed on a weekly basis. Whereas, secondary mice derived from untreated primaries succumbed from ATL between 3 and 4 weeks after transplantation, we observed a biphasic growth intersected by a spontaneous regression phase in untreated secondary mice transplanted from As/IFN treated primary mice. This process was associated with telomere shortening, DNA damage, activation of the PML/P53 axis, and senescence in tumor cells. Interestingly, injection of spleen derived ATL cells from secondary SCID sacrificed before ATL senescence, into untreated tertiary and quaternary SCID, resulted in normal ATL development, indicating that senescence of ATL cells in secondary SCID mice resulted in a change of the microenvironment that culminated in ATL cell apoptosis and loss of ATL LIC activity. Furthermore, injection of spleen derived ATL cells from untreated secondary SCID sacrificed before or after ATL senescence, into untreated tertiary NSG mice, resulted in normal ATL development. These NSG mice, contrary to SCID mice, lack natural killer (NK) cells and have an impaired macrophage activity. In agreement with these results, injection of spleen derived ATL cells from treated primary SCID into untreated secondary NSG mice, resulted in normal ATL development. RT-PCR and ELISA analysis of sorted CD25 positive (ATL) or negative (microenvironment) cells revealed treatment-induced upregulation of MIP1, RANTES, and IFN-γ in the CD25 negative fraction in untreated secondary SCID, following ATL senescence, and concomitant with apoptosis and loss of LIC activity. Conversely, and as previously seen in treated ATL patients, IL-10 expression was rapidly and dramatically downregulated in the CD25 positive fraction in untreated secondary SCID mice. Addition of the proteasome inhibitor PS-341 to AS/IFN treatment of primary mice reverted all above results, presumably indicating the role of As/IFN-induced Tax degradation in the observed phenotypes.Collectively, these results demonstrate that As/IFN-induced, PML/p53 dependent, senescence of ATL cells resulted in activation of the innate immunity that culminated in apoptosis and loss of ATL LIC activity. The striking similarity of these results with patients' data suggests a critical role for innate immunity in ATL cure. DisclosuresHermine:Novartis: Research Funding; Hybrigenics: Research Funding; Celgene: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; INatherys: Equity Ownership, Research Funding.

The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.

Restoration of ceramide de novo synthesis by the synthetic retinoid ST1926 as it induces adult T-cell leukemia cell death.

Ceramide (Cer) is a bioactive cellular lipid with compartmentalized and tightly regulated levels. Distinct metabolic pathways lead to the generation of Cer species with distinguishable roles in oncogenesis. Deregulation of Cer pathways has emerged as an important mechanism for acquired chemotherapeutic resistance. Adult T-cell leukemia (ATL) cells are defective in Cer synthesis. ATL is an aggressive neoplasm that develops following infection with human T-cell lymphotropic virus-1 (HTLV-1) where the viral oncogene Tax contributes to the pathogenesis of the disease. ATL cells, resistant to all-trans-retinoic acid, are sensitive to pharmacologically achievable concentrations of the synthetic retinoid ST1926. We studied the effects of ST1926 on Cer pathways in ATL cells. ST1926 treatment resulted in early Tax oncoprotein degradation in HTLV-1-treated cells. ST1926 induced cell death and a dose- and time-dependent accumulation of Cer in malignant T cells. The kinetics and degree of Cer production showed an early response upon ST1926 treatment. ST1926 enhanced de novo Cer synthesis via activation of ceramide synthase CerS(s) without inhibiting dihydroceramide desaturase, thereby accumulating Cer rather than the less bioactive dihydroceramide. Using labeling experiments with the unnatural 17-carbon sphinganine and measuring the generated Cer species, we showed that ST1926 preferentially induces the activities of a distinct set of CerS(s). We detected a delay in cell death response and interruption of Cer generation in response to ST1926 in Molt-4 cells overexpressing Bcl-2. These results highlight the potential role of ST1926 in inducing Cer levels, thus lowering the threshold for cell death in ATL cells.

A novel positive feedback-loop between the HTLV-1 oncoprotein Tax and NF-\u03baB activity in T-cells

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) infects primarily CD4+ T-lymphocytes and evoques severe diseases, predominantly Adult T-Cell Leukemia/ Lymphoma (ATL/L) and HTLV-1-associated Myelopathy/ Tropical Spastic Paraparesis (HAM/TSP). The viral transactivator of the pX region (Tax) is important for initiating malignant transformation, and deregulation of the major signaling pathway nuclear factor of kappa B (NF-κB) by Tax represents a hallmark of HTLV-1 driven cancer.ResultsHere we found that Tax mutants which are defective in NF-κB signaling showed diminished protein expression levels compared to Tax wildtype in T-cells, whereas Tax transcript levels were comparable. Strikingly, constant activation of NF-κB signaling by the constitutive active mutant of inhibitor of kappa B kinase (IKK2, IKK-β), IKK2-EE, rescued protein expression of the NF-κB defective Tax mutants M22 and K1-10R and even increased protein levels of Tax wildtype in various T-cell lines while Tax transcript levels were only slightly affected. Using several Tax expression constructs, an increase of Tax protein occurred independent of Tax transcripts and independent of the promoter used. Further, Tax and M22 protein expression were strongly enhanced by 12-O-Tetradecanoylphorbol-13-Acetate [TPA; Phorbol 12-myristate 13-acetate (PMA)]/ ionomycin, inducers of NF-κB and cytokine signaling, but not by tumor necrosis factor alpha (TNF-α). On the other hand, co-expression of Tax with a dominant negative inhibitor of κB, IκBα-DN, or specific inhibition of IKK2 by the compound ACHP, led to a vast decrease in Tax protein levels to some extent independent of Tax transcripts in transiently transfected and Tax-transformed T-cells. Cycloheximide chase experiments revealed that co-expression of IKK2-EE prolongs the half-life of M22, and constant repression of NF-κB signaling by IκBα-DN strongly reduces protein stability of Tax wildtype suggesting that NF-κB activity is required for Tax protein stability. Finally, protein expression of Tax and M22 could be recovered by NH4Cl and PYR-41, inhibitors of the lysosome and the ubiquitin-activating enzyme E1, respectively.ConclusionsTogether, these findings suggest that Tax’s capability to induce NF-κB is critical for protein expression and stabilization of Tax itself. Overall, identification of this novel positive feedback loop between Tax and NF-κB in T-cells improves our understanding of Tax-driven transformation.

Mechanisms of Oncogenesis by HTLV-1 Tax.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a neoplasm of CD4+CD25+ T cells that occurs in 2–5% of infected individuals after decades of asymptomatic latent infection. Multiple HTLV-1-encoded regulatory proteins, including Tax and HTLV-1 basic leucine zipper factor (HBZ), play key roles in viral persistence and latency. The HTLV-1 Tax oncoprotein interacts with a plethora of host cellular proteins to regulate viral gene expression and also promote the aberrant activation of signaling pathways such as NF-κB to drive clonal proliferation and survival of T cells bearing the HTLV-1 provirus. Tax undergoes various post-translational modifications such as phosphorylation and ubiquitination that regulate its function and subcellular localization. Tax shuttles in different subcellular compartments for the activation of anti-apoptotic genes and deregulates the cell cycle with the induction of DNA damage for the accumulation of genomic instability that can result in cellular immortalization and malignant transformation. However, Tax is highly immunogenic and therefore HTLV-1 has evolved numerous strategies to tightly regulate Tax expression while maintaining the pool of anti-apoptotic genes through HBZ. In this review, we summarize the key findings on the oncogenic mechanisms used by Tax that set the stage for the development of ATLL, and the strategies used by HTLV-1 to tightly regulate Tax expression for immune evasion and viral persistence.

The effect of alcoholic extract from Eucalyptus camaldulensis leaves on HTLV-1 Tax activities

ABSTRACT HTLV-1 is a human retrovirus responsible for adult T-cell leukemia (ATL) and certain other clinical disorders. The viral Tax oncoprotein plays a central role in HTLV-1 pathogenicity, mainly due to its capacity of inducing the transcriptional activity of various transcriptional factors like NFқB. Eucalyptus camaldulensis (Ec) is considered as a traditional medicinal plant with valuable therapeutic effects. Here we evaluated the activity of its ethanolic leave extract on different Tax activities by testing its influence on Tax-induced activity of NFқB and HTLV-1 LTR in Jurkat cells. Our results showed that Ec inhibited Tax induced activation of NFқB -, SRF- dependent promoters and HTLV-1 LTR. Ec extract has no effect on the binding of Tax to NFқB while it strongly prevented the degradation of IҝBα induced by Tax probably as a result of preventing the link between Tax and IKKγ. In addition, increasing the cellular level of P-TEFb-cyclinT1 significantly reduced the inhibitory effect of Ec on Tax activities, probably by preventing the interaction between Tax and P-TEFb-cyclin T1. The 40%-MeOH fraction of this extract, which is rich with polyphenols, offered the highest inhibitory effect against Tax activities. Further studies are required for the isolation and identification of active component/s in this extract which may be developed in the future as preventive/curing drugs for HTLV-1 related diseases.

Human T-Cell Lymphotropic Virus Type 1 Transactivator Tax Exploits the XPB Subunit of TFIIH during Viral Transcription

Human T-cell lymphotropic virus type 1 (HTLV-1) Tax oncoprotein is required for viral gene expression. Tax transactivates the viral promoter by recruiting specific transcription factors but also by interfering with general transcription factors involved in the preinitiation step, such as TFIIA and TFIID. However, data are lacking regarding Tax interplay with TFIIH, which intervenes during the last step of preinitiation. We previously reported that XPB, the TFIIH subunit responsible for promoter opening and promoter escape, is required for Tat-induced human-immunodeficiency virus promoter transactivation. Here, we investigated whether XPB may also play a role in HTLV-1 transcription. We report that Tax and XPB directly interact in vitro and that endogenous XPB produced by HTLV-1-infected T cells binds to Tax and is recruited on proviral LTRs. In contrast, XPB recruitment at the LTR is not detected in Tax-negative HTLV-1-infected T cells and is strongly reduced when Tax-induced HTLV-1 LTR transactivation is blocked. XPB overexpression does not affect basal HTLV-1 promoter activation but enhances Tax-mediated transactivation in T cells. Conversely, downregulating XPB strongly reduces Tax-mediated transactivation. Importantly, spironolactone (SP)-mediated inhibition of LTR activation can be rescued by overexpressing XPB but not XPD, another TFIIH subunit. Furthermore, an XPB mutant defective for the ATPase activity responsible for promoter opening does not show rescue of the effect of SP. Finally, XPB downregulation reduces viability of Tax-positive but not Tax-negative HTLV-1-transformed T cell lines. These findings reveal that XPB is a novel cellular cofactor hijacked by Tax to facilitate HTLV-1 transcription.IMPORTANCE HTLV-1 is considered the most potent human oncovirus and is also responsible for severe inflammatory disorders. HTLV-1 transcription is undertaken by RNA polymerase II and is controlled by the viral oncoprotein Tax. Tax transactivates the viral promoter first via the recruitment of CREB and its cofactors to the long terminal repeat (LTR). However, how Tax controls subsequent steps of the transcription process remains unclear. In this study, we explore the link between Tax and the XPB subunit of TFIIH that governs, via its ATPase activity, the promoter-opening step of transcription. We demonstrate that XPB is a novel physical and functional partner of Tax, recruited on HTLV-1 LTR, and required for viral transcription. These findings extend the mechanism of Tax transactivation to the recruitment of TFIIH and reinforce the link between XPB and transactivator-induced viral transcription.

EOS, an Ikaros family zinc finger transcription factor, interacts with the HTLV-1 oncoprotein Tax and is downregulated in peripheral blood mononuclear cells of HTLV-1-infected individuals, irrespective of clinical statuses

BackgroundEOS plays an important role in maintaining the suppressive function of regulatory T cells (Tregs), and induces a regulated transformation of Tregs into T helper-like cells, which are capable of secreting proinflammatory cytokines in response to specific inflammatory signals. Meanwhile, significant reduction in Treg activity along with production of proinflammatory cytokines has been reported in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).MethodsIn this study, to examine whether there is an alteration in EOS expression in peripheral blood mononuclear cells (PBMCs) derived from HTLV-1-infected individuals especially HAM/TSP, we investigated the expression of HTLV-1 tax genotype, proviral load (PVL), and the mRNA expression of tax, HBZ and EOS in HTLV-1 infected individuals including adult T-cell leukemia/lymphoma (ATL), HAM/TSP, or asymptomatic carriers. The expression levels of EOS mRNA and protein in various HTLV-1-infected or uninfected human T-cell lines were also investigated.ResultsEOS was highly expressed at the protein level in most HTLV-1 infected T-cell lines, and was augmented after the HTLV-1 regulatory factor Tax was induced in a Tax-inducible JPX-9 cell line. Immunoprecipitation experiments demonstrated a physical interaction between EOS and the viral regulatory protein Tax, but not HBZ. Meanwhile, there was a significant decrease in EOS mRNA levels in PBMCs of HTLV-1 infected individuals irrespective of their clinical statuses. We found an inverse correlation between EOS mRNA levels and HTLV-1 PVL in ATL patients, and positive correlations between both EOS mRNA load and PVL, and EOS and HBZ mRNA load in HAM/TSP patients, whereas this correlation was not observed in other clinical statuses.ConclusionsThese findings suggest that both Tax and HBZ can alter the expression of EOS through undetermined mechanisms, and dysregulated expression of EOS in PBMCs of HTLV-1 infected individuals may contribute to the pathological progression of HTLV-1-associated diseases, such as ATL and HAM/TSP.

SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-\u03baB activation through its ubiquitin binding function

The NF-κB pathway is constitutively activated in adult T cell leukemia, an aggressive malignancy caused by Human T Leukemia Virus type 1 (HTLV-1). The viral oncoprotein Tax triggers this constitutive activation by interacting with the ubiquitin-rich IKK complex. We previously demonstrated that Optineurin and TAX1BP1, two members of the ubiquitin-binding, Sequestosome-1 (SQSTM-1/p62)-like selective autophagy receptor family, are involved in Tax-mediated NF-κB signaling. Here, using a proximity-dependent biotinylation approach (BioID), we identify p62 as a new candidate partner of Tax and confirm the interaction in infected T cells. We then demonstrate that p62 knock-out in MEF cells as well as p62 knock-down in HEK293T cells significantly reduces Tax-mediated NF-κB activity. We further show that although p62 knock-down does not alter NF-κB activation in Jurkat T cells nor in infected T cells, p62 does potentiate Tax-mediated NF-κB activity upon over-expression in Jurkat T cells. We next show that p62 associates with the Tax/IKK signalosome in cells, and identify the 170–206 domain of p62 as sufficient for the direct, ubiquitin-independent interaction with Tax. However, we observe that this domain is dispensable for modulating Tax activity in cells, and functional analysis of p62 mutants indicates that p62 could potentiate Tax activity in cells by facilitating the association of ubiquitin chains with the Tax/IKK signalosome. Altogether, our results identify p62 as a new ubiquitin-dependent modulator of Tax activity on NF-κB, further highlighting the importance of ubiquitin in the signaling activity of the viral Tax oncoprotein.

Collagen IV (COL4A1, COL4A2), a Component of the Viral Biofilm, Is Induced by the HTLV-1 Oncoprotein Tax and Impacts Virus Transmission.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for Adult T-Cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 infects CD4+ T-cells via cell-to-cell transmission requiring reorganization of the cytoskeleton and expression of the viral transactivator and oncoprotein Tax. Viruses spread at the virological synapse (VS), a virus-induced specialized cell-cell contact, by polarized budding into synaptic clefts, and by cell surface transfer of viral biofilms (VBs). Since little is known about Tax’s role in formation of the VB, we asked which component of the VB is regulated by Tax and important for HTLV-1 transmission. Collagens are not only structural proteins of the extracellular matrix and basal membrane but also represent an important component of the VB. Here, we report that among the collagens known to be present in VBs, COL4 is specifically upregulated in the presence of HTLV-1 infection. Further, we found that transient expression of Tax is sufficient to induce COL4A1 and COL4A2 transcripts in Jurkat and CCRF-CEM T-cells, while robust induction of COL4 protein requires continuous Tax expression as shown in Tax-transformed T-cell lines. Repression of Tax led to a significant reduction of COL4A1/A2 transcripts and COL4 protein. Mechanistically, luciferase-based promoter studies indicate that Tax activates the COL4A2 and, to a less extent, the COL4A1 promoter. Imaging showing partial co-localization of COL4 with the viral Gag protein in VBs at the VS and transfer of COL4 and Gag to target cells suggests a role of COL4 in VB formation. Strikingly, in chronically infected C91-PL cells, knockout of COL4A2 impaired Gag transfer between infected T-cells and acceptor T-cells, while release of virus-like particles was unaffected. Taken together, we identified COL4 (COL4A1, COL4A2) as a component of the VB and a novel cellular target of Tax with COL4A2 appearing to impact virus transmission. Thus, this study is the first to provide a link between Tax’s activity and VB formation by hijacking COL4 protein functions.