RESUMEN
An estimated 10-20 million people worldwide are infected with human T cell leukemia virus type 1 (HTLV-1), with endemic areas of infection in Japan, Australia, the Caribbean, and Africa. HTLV-1 is the causative agent of adult T cell leukemia (ATL) and HTLV-1 associated myopathy/tropic spastic paraparesis (HAM/TSP). HTLV-1 expresses several regulatory and accessory genes that function at different stages of the virus life cycle. The regulatory gene Tax-1 is required for efficient virus replication, as it drives transcription of viral gene products, and has also been demonstrated to play a key role in the pathogenesis of the virus. Several studies have identified a PDZ binding motif (PBM) at the carboxyl terminus of Tax-1 and demonstrated the importance of this domain for HTLV-1 induced cellular transformation. Using a mass spectrometry-based proteomics approach we identified sorting nexin 27 (SNX27) as a novel interacting partner of Tax-1. Further, we demonstrated that their interaction is mediated by the Tax-1 PBM and SNX27 PDZ domains. SNX27 has been shown to promote the plasma membrane localization of glucose transport 1 (GLUT1), one of the receptor molecules of the HTLV-1 virus, and the receptor molecule required for HTLV-1 fusion and entry. We postulated that Tax-1 alters GLUT1 localization via its interaction with SNX27. We demonstrate that over expression of Tax-1 in cells causes a reduction of GLUT1 on the plasma membrane. Furthermore, we show that knockdown of SNX27 results in increased virion release and decreased HTLV-1 infectivity. Collectively, we demonstrate the first known mechanism by which HTLV-1 regulates a receptor molecule post-infection.
Asunto(s)
Productos del Gen tax/fisiología , Transportador de Glucosa de Tipo 1/fisiología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Receptores Virales/fisiología , Secuencia de Aminoácidos , Técnicas de Silenciamiento del Gen , Productos del Gen tax/química , Productos del Gen tax/genética , Células HEK293 , Infecciones por HTLV-I/genética , Infecciones por HTLV-I/fisiopatología , Infecciones por HTLV-I/virología , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/fisiología , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Modelos Biológicos , Dominios PDZ , Dominios y Motivos de Interacción de Proteínas , Nexinas de Clasificación/química , Nexinas de Clasificación/genética , Nexinas de Clasificación/fisiología , Virulencia/genética , Virulencia/fisiología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/fisiologíaRESUMEN
The human T-cell leukemia virus-1 (HTLV-1) oncoprotein Tax drives cell proliferation and resistance to apoptosis early in the pathogenesis of adult T-cell leukemia (ATL). Subsequently, probably as a result of specific immunoediting, Tax expression is down-regulated and functionally replaced by somatic driver mutations of the host genome. Both amplification and point mutations of interferon regulatory factor 4 (IRF4) have been previously detected in ATL., K59R is the most common single-nucleotide variation of IRF4 and is found exclusively in ATL. High-throughput whole-exome sequencing revealed recurrent activating genetic alterations in the T-cell receptor, CD28, and NF-κB pathways. We found that IRF4, which is transcriptionally activated downstream of these pathways, is frequently mutated in ATL. IRF4 RNA, protein, and IRF4 transcriptional targets are uniformly elevated in HTLV-1-transformed cells and ATL cell lines, and IRF4 was bound to genomic regulatory DNA of many of these transcriptional targets in HTLV-1-transformed cell lines. We further noted that the K59R IRF4 mutant is expressed at higher levels in the nucleus than WT IRF4 and is transcriptionally more active. Expression of both WT and the K59R mutant of IRF4 from a constitutive promoter in retrovirally transduced murine bone marrow cells increased the abundance of T lymphocytes but not myeloid cells or B lymphocytes in mice. IRF4 may represent a therapeutic target in ATL because ATL cells select for a mutant of IRF4 with higher nuclear expression and transcriptional activity, and overexpression of IRF4 induces the expansion of T lymphocytes in vivo.
Asunto(s)
Factores Reguladores del Interferón/genética , Leucemia-Linfoma de Células T del Adulto/genética , Mutación , Adulto , Animales , Apoptosis , Antígenos CD28/genética , Antígenos CD28/metabolismo , Núcleo Celular/metabolismo , Transformación Celular Viral , Citosol/metabolismo , ADN/metabolismo , Dimerización , Técnicas de Silenciamiento del Gen , Productos del Gen tax/genética , Productos del Gen tax/fisiología , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Factores Reguladores del Interferón/metabolismo , Células Jurkat , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Transcripción Genética , Regulación hacia Arriba , Secuenciación del ExomaAsunto(s)
Acetilglucosamina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación Viral de la Expresión Génica , Productos del Gen tax/fisiología , Procesamiento Proteico-Postraduccional , Transformación Celular Viral/genética , Productos del Gen tax/metabolismo , Glicosilación , Humanos , Unión ProteicaRESUMEN
The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Productos del Gen tax/fisiología , Proteína Quinasa 8 Activada por Mitógenos/fisiología , Proteínas de Neoplasias/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/genética , División Celular , Secuencia de Consenso , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica , Productos del Gen tax/genética , Humanos , Células Jurkat , FN-kappa B/fisiología , Proteínas de Neoplasias/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/fisiología , Transfección , Tretinoina/farmacologíaRESUMEN
The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD4-Positivos/virología , Transformación Celular Viral/fisiología , Factores de Transcripción Forkhead/fisiología , Productos del Gen tax/fisiología , Autofagia/fisiología , Linfocitos T CD4-Positivos/inmunología , Transformación Celular Neoplásica , Transformación Celular Viral/inmunología , Células Cultivadas , Receptores ErbB/fisiología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/patogenicidad , Virus Linfotrópico T Tipo 2 Humano/fisiología , Humanos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/fisiología , Subgrupos de Linfocitos T/virologíaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is a delta-type retrovirus that induces malignant and inflammatory diseases during its long persistence in vivo. HTLV-1 can infect various kinds of cells; however, HTLV-1 provirus is predominantly found in peripheral CD4 T cells in vivo. Here we find that TCF1 and LEF1, two Wnt transcription factors that are specifically expressed in T cells, inhibit viral replication through antagonizing Tax functions. TCF1 and LEF1 can each interact with Tax and inhibit Tax-dependent viral expression and activation of NF-κB and AP-1. As a result, HTLV-1 replication is suppressed in the presence of either TCF1 or LEF1. On the other hand, T-cell activation suppresses the expression of both TCF1 and LEF1, and this suppression enables Tax to function as an activator. We analyzed the thymus of a simian T-cell leukemia virus type 1 (STLV-1) infected Japanese macaque, and found a negative correlation between proviral load and TCF1/LEF1 expression in various T-cell subsets, supporting the idea that TCF1 and LEF1 negatively regulate HTLV-1 replication and the proliferation of infected cells. Thus, this study identified TCF1 and LEF1 as Tax antagonistic factors in vivo, a fact which may critically influence the peripheral T-cell tropism of this virus.
Asunto(s)
Productos del Gen tax/antagonistas & inhibidores , Virus Linfotrópico T Tipo 1 Humano/fisiología , Factor de Unión 1 al Potenciador Linfoide/fisiología , Factor 1 de Transcripción de Linfocitos T/fisiología , Activación Transcripcional/fisiología , Animales , Línea Celular , Regulación hacia Abajo , Productos del Gen tax/fisiología , Humanos , Macaca , Virus Linfotrópico T Tipo 1 de los Simios/aislamiento & purificación , Carga ViralRESUMEN
The mechanisms involved in the persistence of activated CD4+ T lymphocytes following primary human T leukemia/lymphoma virus type 1 (HTLV-1) infection remain unclear. Here, we demonstrate that the HTLV-1 Tax oncoprotein modulates phosphorylation and transcriptional activity of the FOXO3a transcription factor, via upstream activation of the AKT pathway. De novo HTLV-1 infection of CD4+ T cells or direct lentiviral-mediated introduction of Tax led to AKT activation and AKT-dependent inactivation of FOXO3a, via phosphorylation of residues Ser253 and Thr32. Inhibition of FOXO3a signalling led to the long-term survival of a population of highly activated, terminally differentiated CD4+Tax+CD27negCCR7neg T cells that maintained the capacity to disseminate infectious HTLV-1. CD4+ T cell persistence was reversed by chemical inhibition of AKT activity, lentiviral-mediated expression of a dominant-negative form of FOXO3a or by specific small interfering RNA (siRNA)-mediated silencing of FOXO3a. Overall this study provides new mechanistic insight into the strategies used by HTLV-1 to increase long-term maintenance of Tax+CD4+ T lymphocytes during the early stages of HTLV-1 pathogenesis.
Asunto(s)
Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Factores de Transcripción Forkhead/antagonistas & inhibidores , Productos del Gen tax/fisiología , Infecciones por HTLV-I/fisiopatología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Diferenciación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/efectos de los fármacos , Factores de Transcripción Forkhead/fisiología , Infecciones por HTLV-I/patología , Humanos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , ARN Interferente Pequeño/farmacología , Transducción de Señal/fisiología , Proteínas Virales/fisiologíaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion-induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Transformación Celular Viral , Productos del Gen tax/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Interleucina-2/deficiencia , Leucemia-Linfoma de Células T del Adulto/virología , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Linfocitos T/virología , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
One of the hallmarks of cancer, global gene expression alteration, is closely associated with the development and malignant characteristics associated with adult T-cell leukemia (ATL) as well as other cancers. Here, we show that aberrant overexpression of the Ellis Van Creveld (EVC) family is responsible for cellular Hedgehog (HH) activation, which provides the pro-survival ability of ATL cells. Using microarray, quantitative RT-PCR and immunohistochemistry we have demonstrated that EVC is significantly upregulated in ATL and human T-cell leukemia virus type I (HTLV-1)-infected cells. Epigenetic marks, including histone H3 acetylation and Lys4 trimethylation, are specifically accumulated at the EVC locus in ATL samples. The HTLV-1 Tax participates in the coordination of EVC expression in an epigenetic fashion. The treatment of shRNA targeting EVC, as well as the transcription factors for HH signaling, diminishes the HH activation and leads to apoptotic death in ATL cell lines. We also showed that a HH signaling inhibitor, GANT61, induces strong apoptosis in the established ATL cell lines and patient-derived primary ATL cells. Therefore, our data indicate that HH activation is involved in the regulation of leukemic cell survival. The epigenetically deregulated EVC appears to play an important role for HH activation. The possible use of EVC as a specific cell marker and a novel drug target for HTLV-1-infected T-cells is implicated by these findings. The HH inhibitors are suggested as drug candidates for ATL therapy. Our findings also suggest chromatin rearrangement associated with active histone markers in ATL.
Asunto(s)
Epigénesis Genética , Proteínas Hedgehog/metabolismo , Leucemia de Células T/genética , Proteínas/genética , Antineoplásicos/farmacología , Secuencia de Bases , Estudios de Casos y Controles , Supervivencia Celular , Islas de CpG , Metilación de ADN , Regulación Leucémica de la Expresión Génica , Productos del Gen tax/fisiología , Células HEK293 , Humanos , Células Jurkat , Leucemia de Células T/metabolismo , Proteínas de la Membrana , Datos de Secuencia Molecular , Proteínas/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Análisis de Secuencia de ADN , Transducción de Señal , Transcripción GenéticaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Transgenic (Tg) mice expressing HTLV-1 Tax also develop T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. We found that 8 of 297 Tax-Tg mice developed HAM/TSP-like disease with symmetrical paraparesis of the hind limbs, but these symptoms were absent in non-Tg littermates and in other mice strains at our animal facilities. We could perform detailed evaluations for five of these mice. These evaluations showed that the disease was not inflammatory, unlike that in HAM/TSP patients, but instead involved the invasion of histiocytic sarcoma cells into the lumbar spinal cord from the bone marrow where they had undergone extensive proliferation.
Asunto(s)
Neoplasias de la Médula Ósea/patología , Modelos Animales de Enfermedad , Productos del Gen tax/genética , Sarcoma Histiocítico/patología , Paraparesia Espástica Tropical/etiología , Médula Espinal/patología , Animales , Quimiocinas/sangre , Cruzamientos Genéticos , Citocinas/sangre , Femenino , Productos del Gen tax/fisiología , Miembro Posterior/fisiopatología , Sarcoma Histiocítico/complicaciones , Virus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T del Adulto/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Invasividad Neoplásica , Paraparesia Espástica Tropical/patología , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), and the viral oncoprotein Tax plays key roles in the immortalization of human T cells, lifelong persistent infection, and leukemogenesis. We herein identify the ubiquitin-specific protease 10 (USP10) as a Tax-interactor in HTLV-1-infected T cells. USP10 is an antistress factor against various environmental stresses, including viral infections and oxidative stress. On exposure to arsenic, an oxidative stress inducer, USP10 is recruited into stress granules (SGs), and USP10-containing SGs reduce reactive oxygen species (ROS) production and inhibit ROS-dependent apoptosis. We found that interaction of Tax with USP10 inhibits arsenic-induced SG formation, stimulates ROS production, and augments ROS-dependent apoptosis in HTLV-1-infected T cells. These findings suggest that USP10 is a host factor that inhibits stress-induced ROS production and apoptosis in HTLV-1-infected T cells; however, its activities are attenuated by Tax. A clinical study showed that combination therapy containing arsenic is effective against some forms of ATL. Therefore, these findings may be relevant to chemotherapy against ATL.
Asunto(s)
Apoptosis , Productos del Gen tax/fisiología , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/fisiología , Ubiquitina Tiolesterasa/metabolismo , Adulto , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/inmunología , Apoptosis/fisiología , Arsenitos/farmacología , Células Cultivadas , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Células HEK293 , Humanos , Leucemia de Células T/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Unión Proteica/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Transfección , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/fisiologíaRESUMEN
Patients with HIV-1 and human T-lymphotropic virus type 2 (HTLV-2) coinfections often exhibit a clinical course similar to that seen in HIV-1-infected individuals who are long-term nonprogressors. These findings have been attributed in part to the ability of HTLV-2 to activate production of antiviral chemokines and to downregulate the CCR5 coreceptor on lymphocytes. To further investigate these observations, we tested the ability of recombinant Tax1 and Tax2 proteins to suppress HIV-1 viral replication in vitro. R5-tropic HIV-1 (NLAD8)-infected peripheral blood mononuclear cells (PBMCs) were treated daily with recombinant Tax1 and Tax2 proteins (dosage range 1-100 pM). Culture supernatants were collected at intervals from days 1 to 22 postinfection and assayed for levels of HIV-1 p24 antigen by ELISA. Treatment of PBMCs with Tax2 protein resulted in a significant reduction in HIV-1 p24 antigen levels (p<0.05) at days 10, 14, and 18 postinfection compared to HIV-1-infected or mock-treated PBMCs. This was preceded by the detection of increased levels of CC-chemokines MIP-1α/CCL3, MIP-1ß/CCL4, and RANTES/CCL5 on days 1-7 of infection. Similar, but less robust inhibition was observed in Tax1-treated PBMCs. These results support the contention that Tax1 and Tax2 play a role in generating antiviral responses against HIV-1 in vivo and in vitro.
Asunto(s)
Productos del Gen tax/fisiología , VIH-1/fisiología , Infecciones por HTLV-I/complicaciones , Quimiocinas CC/biosíntesis , Coinfección/inmunología , Coinfección/virología , Productos del Gen tax/inmunología , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/complicaciones , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Infecciones por HTLV-I/inmunología , Infecciones por HTLV-I/virología , Infecciones por HTLV-II/complicaciones , Infecciones por HTLV-II/inmunología , Infecciones por HTLV-II/virología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Interferencia Viral/inmunología , Interferencia Viral/fisiología , Replicación Viral/inmunología , Replicación Viral/fisiologíaRESUMEN
BACKGROUND: Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood. RESULTS: Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway. CONCLUSIONS: This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation.
Asunto(s)
Daño del ADN , Reparación del ADN por Unión de Extremidades , Replicación del ADN , Productos del Gen tax/fisiología , Western Blotting , Línea Celular , Ensayo Cometa , Productos del Gen tax/genética , Humanos , Microscopía Fluorescente , FN-kappa B/metabolismoRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), which is an aggressive and fatal CD4(+) T cell malignancy. MicroRNA (miRNA), a novel class of RNA that regulates gene expression, is involved in many cellular processes such as growth, development and apoptosis. It has recently been linked to several cancer phenotypes. However, aberrant miRNA expression and its pathologic significance in ATL are not well documented. Here, we investigated the role of miRNAs in HTLV-1-related leukemogenesis. The results showed that miR-146a was upregulated in HTLV-1-infected T-cell lines compared to uninfected T-cell lines. Tax-induced miR-146a expression in a NF-κB-dependent manner and inhibited the expression of gene harboring the target sequence of miR-146a on its 3'UTR. Inhibition of miR-146a function by anti-miRNA inhibitor reduced the proliferation of HTLV-1-infected T-cell lines but not that of uninfected T-cell lines. Moreover, overexpression of miR-146a enhanced the growth of an HTLV-1-infected T-cell line. Our findings suggest that miR-146a is a potentially suitable therapeutic target of ATL.
Asunto(s)
Productos del Gen tax/fisiología , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T del Adulto/genética , MicroARNs/genética , Linfocitos T/virología , Apoptosis , Línea Celular Tumoral , Humanos , Leucemia-Linfoma de Células T del Adulto/virología , MicroARNs/metabolismo , MicroARNs/fisiología , FN-kappa B/metabolismo , Linfocitos T/metabolismo , Linfocitos T/patología , Activación TranscripcionalRESUMEN
Ras signaling pathways play an important role in cellular proliferation and survival, and inappropriate activation of Ras frequently results in cell transformation and cancer. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the etiological agent of the adult T-cell leukemia/lymphoma (ATLL), a severe malignancy that has a poor prognosis and exhibits resistance to conventional chemotherapy. Although the mechanisms involved in cell transformation by HTLV-1 have not been completely clarified, it is generally thought that Tax plays a pivotal role in the process. We have previously proposed that a functionally active Ras protein is needed for efficient anti-apoptotic activity of Tax. In this study we report data indicating that the apoptotic resistance of cells expressing Tax, constitutively or transiently, is linked to the intracellular levels of Ras-GTP. Indeed, we found that Tax-positive cells have a high content of active Ras, and that inhibition of Ras signaling, using the antagonist farnesyl thyosalicylic acid (FTS), increases their sensitivity to apoptosis. FTS treatment was also accompanied by a decrease in ERK, but not Akt, phosphorylation. Thus, all together our data suggest that the interaction between Tax and Ras could be important to ATLL pathogenesis, and indicate Ras as a possible target for therapeutic intervention in ATLL patients.
Asunto(s)
Apoptosis/fisiología , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transformación Celular Neoplásica , Células Cultivadas , Productos del Gen tax/fisiología , Humanos , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)-modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB-mediated and decreased cAMP Response Element-Binding (CREB)-mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage-induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes.
Asunto(s)
Productos del Gen tax/metabolismo , Productos del Gen tax/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Daño del ADN/genética , Daño del ADN/fisiología , Expresión Génica/fisiología , Productos del Gen tax/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/fisiología , Unión Proteica , Transporte de Proteínas , Proteína SUMO-1/metabolismo , Distribución Tisular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Ubiquitinación/fisiologíaRESUMEN
Human T cell leukemia virus type-1 (HTLV-1) is the causative agent of a fatal adult T-cell leukemia. Through deregulation of multiple cellular signaling pathways the viral Tax protein has a pivotal role in T-cell transformation. In response to stressful stimuli, cells mount a cellular stress response to limit the damage that environmental forces inflict on DNA or proteins. During stress response, cells postpone the translation of most cellular mRNAs, which are gathered into cytoplasmic mRNA-silencing foci called stress granules (SGs) and allocate their available resources towards the production of dedicated stress-management proteins. Here we demonstrate that Tax controls the formation of SGs and interferes with the cellular stress response pathway. In agreement with previous reports, we observed that Tax relocates from the nucleus to the cytoplasm in response to environmental stress. We found that the presence of Tax in the cytoplasm of stressed cells prevents the formation of SGs and counteracts the shutoff of specific host proteins. Unexpectedly, nuclear localization of Tax promotes spontaneous aggregation of SGs, even in the absence of stress. Mutant analysis revealed that the SG inhibitory capacity of Tax is independent of its transcriptional abilities but relies on its interaction with histone deacetylase 6, a critical component of SGs. Importantly, the stress-protective effect of Tax was also observed in the context of HTLV-1 infected cells, which were shown to be less prone to form SGs and undergo apoptosis under arsenite exposure. These observations identify Tax as the first virally encoded inhibitory component of SGs and unravel a new strategy developed by HTLV-1 to deregulate normal cell processes. We postulate that inhibition of the stress response pathway by Tax would favor cell survival under stressful conditions and may have an important role in HTLV-1-induced cellular transformation.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Productos del Gen tax/fisiología , Histona Desacetilasas/fisiología , ARN Mensajero/metabolismo , Estrés Fisiológico , Células HeLa , Histona Desacetilasa 6 , HumanosRESUMEN
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 cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-ß expression accompanied by reduced HTLV-1 replication and p19(Gag) levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.
Asunto(s)
Productos del Gen tax/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Inmunidad Innata/fisiología , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Secuencia de Bases , Línea Celular , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 Supresora de la Señalización de Citocinas , Técnicas del Sistema de Dos Híbridos , Replicación ViralRESUMEN
The human cytomegalovirus (CMV) major immediate-early (MIE) promoter is widely used in mammalian cells for production of recombinant proteins. It is of great interest to further enhance protein production driven by the CMV promoter. Here, we report that the Tax protein of human T-lymphotropic virus stimulates the transgene expression under the control of CMV MIE promoter in HEK293 cells. At least threefold increases in transient production of recombinant proteins, including luciferase and two biopharmaceutical proteins (erythropoietin and interferon-γ), were detected. Furthermore, cyclic adenosine monophosphate (AMP)-response element binding protein 2 (CREB2) was identified as a cellular cofactor, which might be responsible for Tax transactivation of the CMV MIE promoter. Our results not only demonstrate the potential use of this novel expression strategy for improvement of recombinant protein production in HEK293 cells but also provide the molecular mechanism for Tax-mediated activation of CMV MIE promoter.