RESUMEN
The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) - a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling - to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Regulación hacia Abajo/inmunología , Inestabilidad Genómica/inmunología , Infecciones por HTLV-I/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Leucemia-Linfoma de Células T del Adulto/inmunología , Proteínas de Neoplasias/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Reparación del ADN/inmunología , Proteínas de Unión al ADN/genética , Productos del Gen tax/genética , Productos del Gen tax/inmunología , Infecciones por HTLV-I/genética , Infecciones por HTLV-I/patología , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas de Neoplasias/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFß-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Productos del Gen tax/metabolismo , Transducción de Señal/fisiología , Línea Celular , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Quinasa I-kappa B/metabolismo , Immunoblotting , Quinasas Quinasa Quinasa PAM/metabolismo , Reacción en Cadena de la Polimerasa , Transfección , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Human T lymphotropic virus type I (HTLV-I) infection is largely latent in infected persons. How HTLV-1 establishes latency and reactivates is unclear. Here we show that most HTLV-1-infected HeLa cells become senescent. By contrast, when NF-κB activity is blocked, senescence is averted, and infected cells continue to divide and chronically produce viral proteins. A small population of infected NF-κB-normal HeLa cells expresses low but detectable levels of Tax and Rex, albeit not Gag or Env. In these "latently" infected cells, HTLV-1 LTR trans-activation by Tax persists, but NF-κB trans-activation is attenuated due to inhibition by HBZ, the HTLV-1 antisense protein. Furthermore, Gag-Pol mRNA localizes primarily in the nuclei of these cells. Importantly, HBZ was found to inhibit Rex-mediated export of intron-containing mRNAs. Over-expression of Rex or shRNA-mediated silencing of HBZ led to viral reactivation. Importantly, strong NF-κB inhibition also reactivates HTLV-1. Hence, during HTLV-1 infection, when Tax/Rex expression is robust and dominant over HBZ, productive infection ensues with expression of structural proteins and NF-κB hyper-activation, which induces senescence. When Tax/Rex expression is muted and HBZ is dominant, latent infection is established with expression of regulatory (Tax/Rex/HBZ) but not structural proteins. HBZ maintains viral latency by down-regulating Tax-induced NF-κB activation and senescence, and by inhibiting Rex-mediated expression of viral structural proteins.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Productos del Gen rex/metabolismo , Infecciones por HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Activación Transcripcional/fisiología , Proteínas Virales/metabolismo , Latencia del Virus/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virología , Productos del Gen rex/genética , Productos del Gen tax , Infecciones por HTLV-I/genética , Células HeLa , Humanos , ARN Viral/biosíntesis , ARN Viral/genética , Proteínas de los Retroviridae , Proteínas Virales/genéticaRESUMEN
The HTLV-1 oncoprotein Tax is a potent activator of classical and alternative NF-κB pathways and is thought to promote cell proliferation and transformation via NF-κB activation. We showed recently that hyperactivation of NF-κB by Tax triggers a cellular senescence response (H. Zhi et al., PLoS Pathog. 7:e1002025, 2011). Inhibition of NF-κB activation by expression of I-κBα superrepressor or by small hairpin RNA (shRNA)-mediated knockdown of p65/RelA rescues cells from Tax-induced rapid senescence (Tax-IRS). Here we demonstrate that Tax-IRS is driven by the transcriptional activity of NF-κB. Knockdown of IKKγ, the primary Tax target, by shRNAs abrogated Tax-mediated activation of both classical and alternative NF-κB pathways and rendered knockdown cells resistant to Tax-IRS. Consistent with a critical role of IKKα in the transcriptional activity of NF-κB, IKKα deficiency drastically decreased NF-κB trans-activation by Tax, although it only modestly reduced Tax-mediated I-κBα degradation and NF-κB nuclear localization. In contrast, although IKKß knockdown attenuated Tax-induced NF-κB transcriptional activation, the residual NF-κB activation in IKKß-deficient cells was sufficient to trigger Tax-IRS. Importantly, the phenotypes of NIK and TAK1 knockdown were similar to those of IKKα and IKKß knockdown, respectively. Finally, double knockdown of RelB and p100 had a minor effect on senescence induction by Tax. These data suggest that Tax, through its interaction with IKKγ, helps recruit NIK and TAK1 for IKKα and IKKß activation, respectively. In the presence of Tax, the delineation between the classical and alternative NF-κB pathways becomes obscured. The senescence checkpoint triggered by Tax is driven by the transcriptional activity of NF-κB, which depends on activated IKKα and p65/RelA.
Asunto(s)
Senescencia Celular , Productos del Gen tax/metabolismo , Infecciones por HTLV-I/fisiopatología , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Quinasa I-kappa B/genética , FN-kappa B/metabolismo , Factor de Transcripción ReIA/genética , Activación Transcripcional , Línea Celular , Activación Enzimática , Regulación de la Expresión Génica , Productos del Gen tax/genética , Infecciones por HTLV-I/genética , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Quinasa I-kappa B/metabolismo , FN-kappa B/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27(KIP1) protein and p21(CIP1/WAF1) mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Senescencia Celular/genética , Productos del Gen tax/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , FN-kappa B/metabolismo , Proteínas Virales/fisiología , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Regulación hacia Abajo , Productos del Gen tax/genética , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , ARN Mensajero/metabolismo , Proteínas de los Retroviridae , Replicación Viral/efectos de los fármacosRESUMEN
Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4(+) T cells whose etiology is thought to be associated with the viral trans-activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27(Kip1) and p21(CIP1/WAF1) through protein stabilization and mRNA trans-activation and stabilization, respectively. The Tax-induced surge in p21(CIP1/WAF1) and p27(Kip1) begins in S phase and results in cellular senescence. Importantly, HeLa and SupT1 T cells infected by HTLV-1 also arrest in senescence, thus challenging the notion that HTLV-1 infection causes cell proliferation. Here we use time-lapse microscopy to investigate the effect of Tax on cell cycle progression in two reporter cell lines, HeLa/18x21-EGFP and HeLa-FUCCI, that express enhanced green fluorescent protein (EGFP) under the control of 18 copies of the Tax-responsive 21-bp repeat element and fluorescent ubiquitin cell cycle indicators, respectively. Tax-expressing HeLa cells exhibit elongated or stalled cell cycle phases. Many of them bypass mitosis and become single senescent cells as evidenced by the expression of senescence-associated ß-galactosidase. Such cells have twice the normal equivalent of cellular contents and hence are enlarged, with exaggerated nuclei. Interestingly, nocodazole treatment revealed a small variant population of HeLa/18x21-EGFP cells that could progress into mitosis normally with high levels of Tax expression, suggesting that genetic or epigenetic changes that prevent Tax-induced senescence can occur spontaneously at a detectable frequency.
Asunto(s)
Ciclo Celular/fisiología , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Fusión Artificial Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía por VideoRESUMEN
The immunoproteasome, having been linked to neurodegenerative diseases and hematological cancers, has been shown to play an important role in MHC class I antigen presentation. However, its other pathophysiological functions are still not very well understood. This can be attributed mainly to a lack of appropriate molecular probes that can selectively modulate the immunoproteasome catalytic subunits. Herein, we report the development of molecular probes that selectively inhibit the major catalytic subunit, LMP2, of the immunoproteasome. We show that these compounds irreversibly modify the LMP2 subunit with high specificity. Importantly, LMP2-rich cancer cells compared to LMP2-deficient cancer cells are more sensitive to growth inhibition by the LMP2-specific inhibitor, implicating an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2.