RESUMO
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.
Assuntos
Produtos do Gene tax/fisiologia , Transportador de Glucose Tipo 1/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Receptores Virais/fisiologia , Sequência de Aminoácidos , Técnicas de Silenciamento de Genes , Produtos do Gene tax/química , Produtos do Gene tax/genética , Células HEK293 , Infecções por HTLV-I/genética , Infecções por HTLV-I/fisiopatologia , Infecções por HTLV-I/virologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Modelos Biológicos , Domínios PDZ , Domínios e Motivos de Interação entre Proteínas , Nexinas de Classificação/química , Nexinas de Classificação/genética , Nexinas de Classificação/fisiologia , Virulência/genética , Virulência/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
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.
Assuntos
Fatores Reguladores de Interferon/genética , Leucemia-Linfoma de Células T do Adulto/genética , Mutação , Adulto , Animais , Apoptose , Antígenos CD28/genética , Antígenos CD28/metabolismo , Núcleo Celular/metabolismo , Transformação Celular Viral , Citosol/metabolismo , DNA/metabolismo , Dimerização , Técnicas de Silenciamento de Genes , Produtos do Gene tax/genética , Produtos do Gene tax/fisiologia , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Fatores Reguladores de Interferon/metabolismo , Células Jurkat , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Transcrição Gênica , Regulação para Cima , Sequenciamento do ExomaAssuntos
Acetilglucosamina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene tax/fisiologia , Processamento de Proteína Pós-Traducional , Transformação Celular Viral/genética , Produtos do Gene tax/metabolismo , Glicosilação , Humanos , Ligação ProteicaRESUMO
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.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Produtos do Gene tax/fisiologia , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Proteínas de Neoplasias/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Divisão Celular , Sequência Consenso , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Produtos do Gene tax/genética , Humanos , Células Jurkat , NF-kappa B/fisiologia , Proteínas de Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/fisiologia , Transfecção , Tretinoína/farmacologiaRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Transformação Celular Viral/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Produtos do Gene tax/fisiologia , Autofagia/fisiologia , Linfócitos T CD4-Positivos/imunologia , Transformação Celular Neoplásica , Transformação Celular Viral/imunologia , Células Cultivadas , Receptores ErbB/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírus Linfotrópico T Tipo 2 Humano/patogenicidade , Vírus Linfotrópico T Tipo 2 Humano/fisiologia , Humanos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/virologiaRESUMO
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.
Assuntos
Produtos do Gene tax/antagonistas & inibidores , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Fator 1 de Ligação ao Facilitador Linfoide/fisiologia , Fator 1 de Transcrição de Linfócitos T/fisiologia , Ativação Transcricional/fisiologia , Animais , Linhagem Celular , Regulação para Baixo , Produtos do Gene tax/fisiologia , Humanos , Macaca , Vírus Linfotrópico T Tipo 1 de Símios/isolamento & purificação , Carga ViralRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Fatores de Transcrição Forkhead/antagonistas & inibidores , Produtos do Gene tax/fisiologia , Infecções por HTLV-I/fisiopatologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Diferenciação Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/efeitos dos fármacos , Fatores de Transcrição Forkhead/fisiologia , Infecções por HTLV-I/patologia , Humanos , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/fisiologia , Proteínas Virais/fisiologiaRESUMO
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.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Transformação Celular Viral , Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Interleucina-2/deficiência , Leucemia-Linfoma de Células T do Adulto/virologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/virologia , Animais , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Regulação para Baixo , Técnicas de Silenciamento de Genes , Produtos do Gene tax/genética , Produtos do Gene tax/metabolismo , Células HEK293 , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
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.
Assuntos
Epigênese Genética , Proteínas Hedgehog/metabolismo , Leucemia de Células T/genética , Proteínas/genética , Antineoplásicos/farmacologia , Sequência de Bases , Estudos de Casos e Controles , Sobrevivência Celular , Ilhas de CpG , Metilação de DNA , Regulação Leucêmica da Expressão Gênica , Produtos do Gene tax/fisiologia , Células HEK293 , Humanos , Células Jurkat , Leucemia de Células T/metabolismo , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Análise de Sequência de DNA , Transdução de Sinais , Transcrição GênicaRESUMO
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.
Assuntos
Neoplasias da Medula Óssea/patologia , Modelos Animais de Doenças , Produtos do Gene tax/genética , Sarcoma Histiocítico/patologia , Paraparesia Espástica Tropical/etiologia , Medula Espinal/patologia , Animais , Quimiocinas/sangue , Cruzamentos Genéticos , Citocinas/sangue , Feminino , Produtos do Gene tax/fisiologia , Membro Posterior/fisiopatologia , Sarcoma Histiocítico/complicações , Vírus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T do Adulto/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Invasividade Neoplásica , Paraparesia Espástica Tropical/patologia , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
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.
Assuntos
Apoptose , Produtos do Gene tax/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/fisiologia , Ubiquitina Tiolesterase/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Apoptose/fisiologia , Arsenitos/farmacologia , Células Cultivadas , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Produtos do Gene tax/genética , Produtos do Gene tax/metabolismo , Células HEK293 , Humanos , Leucemia de Células T/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Ligação Proteica/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transfecção , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/fisiologiaRESUMO
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.
Assuntos
Produtos do Gene tax/fisiologia , HIV-1/fisiologia , Infecções por HTLV-I/complicações , Quimiocinas CC/biossíntese , Coinfecção/imunologia , Coinfecção/virologia , Produtos do Gene tax/imunologia , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/complicações , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Infecções por HTLV-II/complicações , Infecções por HTLV-II/imunologia , Infecções por HTLV-II/virologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Interferência Viral/imunologia , Interferência Viral/fisiologia , Replicação Viral/imunologia , Replicação Viral/fisiologiaRESUMO
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.
Assuntos
Dano ao DNA , Reparo do DNA por Junção de Extremidades , Replicação do DNA , Produtos do Gene tax/fisiologia , Western Blotting , Linhagem Celular , Ensaio Cometa , Produtos do Gene tax/genética , Humanos , Microscopia de Fluorescência , NF-kappa B/metabolismoRESUMO
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.
Assuntos
Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T do Adulto/genética , MicroRNAs/genética , Linfócitos T/virologia , Apoptose , Linhagem Celular Tumoral , Humanos , Leucemia-Linfoma de Células T do Adulto/virologia , MicroRNAs/metabolismo , MicroRNAs/fisiologia , NF-kappa B/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Ativação TranscricionalRESUMO
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.
Assuntos
Apoptose/fisiologia , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transformação Celular Neoplásica , Células Cultivadas , Produtos do Gene tax/fisiologia , Humanos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Produtos do Gene tax/metabolismo , Produtos do Gene tax/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Dano ao DNA/genética , Dano ao DNA/fisiologia , Expressão Gênica/fisiologia , Produtos do Gene tax/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/fisiologia , Ligação Proteica , Transporte Proteico , Proteína SUMO-1/metabolismo , Distribuição Tecidual , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Ubiquitinação/fisiologiaRESUMO
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.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Produtos do Gene tax/fisiologia , Histona Desacetilases/fisiologia , RNA Mensageiro/metabolismo , Estresse Fisiológico , Células HeLa , Desacetilase 6 de Histona , HumanosRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Senescência Celular/genética , Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , NF-kappa B/metabolismo , Proteínas Virais/fisiologia , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Regulação para Baixo , Produtos do Gene tax/genética , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , RNA Mensageiro/metabolismo , Proteínas dos Retroviridae , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Imunidade Inata/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/biossíntese , Sequência de Bases , Linhagem Celular , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Supressora da Sinalização de Citocina , Técnicas do Sistema de Duplo-Híbrido , Replicação ViralRESUMO
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.