RESUMEN
Articular cartilage (AC) has poor capacities of regeneration and lesions often lead to osteoarthritis. Current AC reconstruction implies autologous chondrocyte implantation which requires tissue sampling and grafting. An alternative approach would be to use scaffolds containing off-the-shelf allogeneic human articular chondrocytes (HACs). To investigate tolerance of allogeneic HACs by the human immune system, we developed a humanized mouse model implanted with allogeneic cartilage constructs generated in vitro. A prerequisite of the study was to identify a scaffold that would not provoke inflammatory reaction in host. Therefore, we first compared the response of hu-mice to two biomaterials used in regenerative medicine, collagen sponge and agarose hydrogel. Four weeks after implantation in hu-mice, acellular collagen sponges, but not acellular agarose hydrogels, showed positive staining for CD3 (T lymphocytes) and CD68 (macrophages), suggesting that collagen scaffold elicits weak inflammatory reaction. These data led us to deepen our evaluation of the biocompatibility of allogeneic tissue-engineered cartilage by using agarose as scaffold. Agarose hydrogels were combined with allogeneic HACs to reconstruct cartilage in vitro. Particular attention was paid to HLA-A2 compatibility between HACs to be grafted and immune human cells of hu-mice: HLA-A2+ or HLA-A2- HACs agarose hydrogels were cultured in the presence of a chondrogenic cocktail and implanted in HLA-A2+ hu-mice. After four weeks implantation and regardless of the HLA-A2 phenotype, chondrocytes were well-differentiated and produced cartilage matrix in agarose. In addition, no sign of T-cell or macrophage infiltration was seen in the cartilaginous constructs and no significant increase in subpopulations of T lymphocytes and monocytes was detected in peripheral blood and spleen. We show for the first time that humanized mouse represents a useful model to investigate human immune responsiveness to tissue-engineered cartilage and our data together indicate that allogeneic cartilage constructs can be suitable for cartilage engineering.
Asunto(s)
Cartílago Articular/citología , Condrocitos/citología , Condrogénesis , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Osteoartritis/terapia , Trasplante HomólogoRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization.
Asunto(s)
Proliferación Celular , Transformación Celular Viral , Modelos Animales de Enfermedad , Productos del Gen tax/metabolismo , Infecciones por HTLV-I/patología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Linfocitos T/patología , Animales , Femenino , Perfilación de la Expresión Génica , Productos del Gen tax/genética , Células HEK293 , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/virología , Humanos , Activación de Linfocitos , Masculino , Ratones , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Dominios PDZ , Unión Proteica , Linfocitos T/metabolismoRESUMEN
The human T-lymphotropic virus type 1 (HTLV-1) is efficiently transmitted through cellular contacts. While the molecular mechanisms of viral cell-to-cell propagation have been extensively studied in vitro, those facilitating the encounter between infected and target cells remain unknown. In this study, we demonstrate that HTLV-1-infected CD4 T cells secrete a potent chemoattractant, leukotriene B4 (LTB4). LTB4 secretion is dependent on Tax-induced transactivation of the pla2g4c gene, which encodes the cytosolic phospholipase A2 gamma. Inhibition of LTB4 secretion or LTB4 receptor knockdown on target cells reduces T-cell recruitment, cellular contact formation and virus propagation in vitro. Finally, blocking the synthesis of LTB4 in a humanized mouse model of HTLV-1 infection significantly reduces proviral load. This results from a decrease in the number of infected clones while their expansion is not impaired. This study shows the critical role of LTB4 secretion in HTLV-1 transmission both in vitro and in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Leucotrieno B4/metabolismo , Animales , Linfocitos T CD4-Positivos/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Fosfolipasas A2 Grupo IV/genética , Fosfolipasas A2 Grupo IV/metabolismo , Infecciones por HTLV-I/tratamiento farmacológico , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/virología , Interacciones Huésped-Patógeno , Humanos , Indoles/farmacología , Recién Nacido , Células Jurkat , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Ratones Mutantes , FN-kappa B/genética , FN-kappa B/metabolismoRESUMEN
The first discovered human retrovirus, Human T-Lymphotropic Virus type 1 (HTLV-1), is responsible for an aggressive form of T cell leukemia/lymphoma. Mouse models recapitulating the leukemogenesis process have been helpful for understanding the mechanisms underlying the pathogenesis of this retroviral-induced disease. This review will focus on the recent advances in the generation of immunodeficient and human hemato-lymphoid system mice with a particular emphasis on the development of mouse models for HTLV-1-mediated pathogenesis, their present limitations and the challenges yet to be addressed.
Asunto(s)
Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Leucemia-Linfoma de Células T del Adulto/patología , Leucemia-Linfoma de Células T del Adulto/virología , Animales , Humanos , Ratones , Ratones SCIDRESUMEN
UNLABELLED: Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE: Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.
Asunto(s)
Perfilación de la Expresión Génica , Virus Linfotrópico T Tipo 3 de los Simios/genética , Virus Linfotrópico T Tipo 3 de los Simios/fisiología , Proteínas Virales/análisis , Proteínas Virales/genética , Animales , Línea Celular , Núcleo Celular/química , Citosol/química , Humanos , Peso Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/químicaRESUMEN
Human T-cell leukemia virus types 3 and 4 (HTLV-3 and HTLV-4) are recently isolated retroviruses. We have previously characterized HTLV-3- and HTLV-4-encoded antisense genes, termed APH-3 and APH-4, respectively, which, in contrast to HBZ, the HTLV-1 homologue, do not contain a typical bZIP domain (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). As HBZ differentially modulates the transactivation potential of various Jun family members, the effect of APH-3 and APH-4 on JunD-, c-Jun-, and JunB-mediated transcriptional activation was investigated. We first showed that APH-3 and APH-4 upregulated the transactivation potential of all tested Jun family members. Using an human telomerase catalytic subunit (hTERT) promoter construct, our results also highlighted that, unlike HBZ, which solely modulates hTERT expression via JunD, both APH-3 and APH-4 acted positively on the transactivation of the hTERT promoter mediated by tested Jun factors. Coimmunoprecipitation experiments demonstrated that these Jun proteins interacted with APH-3 and APH-4. Although no activation domain was identified for APH proteins, the activation domain of c-Jun was very important in the observed upregulation of its activation potential. We further showed that APH-3 and APH-4 required their putative bZIP-like domains and corresponding leucine residues for interaction and modulation of the transactivation potential of Jun factors. Our results demonstrate that HTLV-encoded antisense proteins behave differently, and that the bZIP-like domains of both APH-3 and APH-4 have retained their interaction potential for Jun members. These studies are important in assessing the differences between HBZ and other antisense proteins, which might further contribute to determining the role of HBZ in HTLV-1-associated diseases. IMPORTANCE HBZ, the antisense transcript-encoded protein from HTLV-1, is now well recognized as a potential factor for adult T-cell leukemia/lymphoma development. In order to better appreciate the mechanism of action of HBZ, comparison to antisense proteins from other HTLV viruses is important. Little is known in relation to the seemingly nonpathogenic HTLV-3 and HTLV-4 viruses, and studies of their antisense proteins are limited to our previously reported study (M. Larocque É Halin, S. Landry, S. J. Marriott, W. M. Switzer, and B. Barbeau, J. Virol. 85:12673-12685, 2011, doi:10.1128/JVI.05296-11). Here, we demonstrate that Jun transcription factors are differently affected by APH-3 and APH-4 compared to HBZ. These intriguing findings suggest that these proteins act differently on viral replication but also on cellular gene expression, and that highlighting their differences of action might lead to important information allowing us to understand the link between HTLV-1 HBZ and ATL in infected individuals.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN sin Sentido/genética , Virus Linfotrópico T Tipo 3 Humano/genética , Virus Linfotrópico T Tipo 3 Humano/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Activación Transcripcional/genética , Animales , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Deltaretrovirus/genética , Deltaretrovirus/metabolismo , Células HEK293 , Células HeLa , Humanos , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-jun/genética , Telomerasa/genética , Telomerasa/metabolismo , Transcripción Genética/genética , Regulación hacia Arriba/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Human T lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). ATLL is a severe malignancy with no effective treatment. HTLV-1 regulatory proteins Tax and HTLV-1 basic leucine zipper factor (HBZ) play a major role in ATLL development, by interfering with cellular functions such as CD4(+) T-cell survival. In this study, we observed that the expression of Bfl-1, an antiapoptotic protein of the Bcl-2 family, is restricted to HTLV-1-infected T-cell lines and to T-cells expressing both Tax and HBZ proteins. We showed that Tax-induced bfl-1 transcription through the canonical NF-κB pathway. Moreover, we demonstrated that Tax cooperated with c-Jun or JunD, but not JunB, transcription factors of the AP-1 family to stimulate bfl-1 gene activation. By contrast, HBZ inhibited c-Jun-induced bfl-1 gene activation, whereas it increased JunD-induced bfl-1 gene activation. We identified one NF-κB, targeted by RelA, c-Rel, RelB, p105/p50, and p100/p52, and two AP-1, targeted by both c-Jun and JunD, binding sites in the bfl-1 promoter of T-cells expressing both Tax and HBZ. Analyzing the potential role of antiapoptotic Bcl-2 proteins in HTLV-1-infected T-cell survival, we demonstrated that these cells are differentially sensitive to silencing of Bfl-1, Bcl-x(L), and Bcl-2. Indeed, both Bfl-1 and Bcl-x(L) knockdowns decreased the survival of HTLV-1-infected T-cell lines, although no cell death was observed after Bcl-2 knockdown. Furthermore, we demonstrated that Bfl-1 knockdown sensitizes HTLV-1-infected T-cells to ABT-737 or etoposide treatment. Our results directly implicate Bfl-1 and Bcl-x(L) in HTLV-1-infected T-cell survival and suggest that both Bfl-1 and Bcl-x(L) represent potential therapeutic targets for ATLL treatment.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T del Adulto/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Adulto , Antineoplásicos Fitogénicos/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Compuestos de Bifenilo/farmacología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Supervivencia Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Etopósido/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Productos del Gen tax/genética , Genes jun/genética , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia-Linfoma de Células T del Adulto/dietoterapia , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Ligasas/genética , Ligasas/metabolismo , Masculino , Antígenos de Histocompatibilidad Menor , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Nitrofenoles/farmacología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-rel , Proteínas de los Retroviridae , Sulfonamidas/farmacología , Factor de Transcripción ReIB/genética , Factor de Transcripción ReIB/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/biosíntesis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células HEK293 , Humanos , Células Jurkat , ARN Helicasas , Proteínas de los Retroviridae , Linfocitos T/metabolismo , Linfocitos T/virología , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
The study of human pathologies is often limited by the absence of animal models which are robust, cost-effective and reproduce the hallmarks of human infections. While mice have been frequently employed to study human diseases, many of important pathogens display unique human tropism. These last two decades the graft of human progenitor cells or tissues into -immunodeficient mice has allowed the elaboration of so called humanized mice. Humanized mouse technology has made rapid progress, and it is now possible to achieve high levels of human chimerism in various organs and tissues, particularly the immune system and the liver. The review briefly summarizes the different models of humanized mice available for in vivo experiments. With a focus on lymphotropic, monocytotropic and hepatotropic viruses, we here discuss the current status and future prospects of these models for studying the pathogenesis of infectious diseases. Furthermore, they provide a powerful tool for the development of innovative therapies.
Asunto(s)
Quimera , Enfermedades Transmisibles , Modelos Animales de Enfermedad , Ratones Mutantes , Animales , Quimera/genética , Quimera/inmunología , Cruzamientos Genéticos , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Predicción , Hepatocitos/trasplante , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Hígado/embriología , Trasplante de Hígado , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes/genética , Ratones Mutantes/inmunología , Quimera por Radiación , Especificidad de la Especie , Timo/embriología , Timo/trasplante , Trasplante Heterólogo , Virosis/tratamiento farmacológico , Virosis/inmunología , Virosis/virologíaRESUMEN
Adult T-cell leukemia (ATL) is an often fatal leukemia of CD4+ T lymphocytes associated with a complex retrovirus, human T-cell leukemia virus type 1 (HTLV-1). Although the viral Tax protein is involved in the proliferation of infected cells during the preleukemic stages, Tax expression is not systematically detected in primary leukemic cells. In 2002, we described the characterization of a novel viral protein that we have termed HBZ for HTLV-1 bZIP factor. This viral factor is encoded on the antisense strand of HTLV-1 proviral DNA, demonstrating the existence of antisense transcription from a promoter located in the 3' LTR. HBZ can negatively control the expression of the other viral proteins by blocking the interaction between Tax and ATF/CREB factors and the recruitment of CBP/p300 by Tax on the promoter. Moreover, recent studies found that the viral HBZ gene was always expressed in leukemic cells, suggesting its involvement in the progression of the infected cells towards malignancy.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Transformación Celular Viral/genética , Regulación Viral de la Expresión Génica , Virus Linfotrópico T Tipo 1 Humano/fisiología , Leucemia-Linfoma de Células T del Adulto/virología , Proteínas Virales/fisiología , Factores de Transcripción Activadores/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , División Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Productos del Gen tax/metabolismo , Genes pX , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Leucina Zippers/genética , Leucina Zippers/fisiología , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/inmunología , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Provirus/genética , ARN Interferente Pequeño/farmacología , Conejos , Proteínas de los Retroviridae , Secuencias Repetidas Terminales/genética , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Proteínas Virales/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Caveolin-1 is implicated in the regulation of signal pathways. Adult T-cell leukemia (ATL) is a T-cell malignancy causatively associated with human T-cell leukemia virus type 1 (HTLV-1). To determine the role of caveolin-1 in leukemogenesis, we examined caveolin-1 expression levels in HTLV-1-infected T-cell lines and ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Caveolin-1-positive ATL cells were detected in ATL lymph nodes and skin lesions, and caveolin-1 was also detected in the plasma of patients with ATL. Infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 expression. The viral protein Tax transcriptionally activated caveolin-1 gene through nuclear factor-kappaB and cAMP response element binding protein signal pathways. HTLV-1-infected T-cell lines, and ATL cells are known to be resistant to transforming growth factor beta (TGF-beta)-induced growth inhibition. Caveolin-1 was colocalized with TGF-beta type I receptor in HTLV-1-infected T-cell lines and suppressed TGF-beta signaling. Caveolin-1 knockdown in an HTLV-1-infected T-cell line exhibited susceptibility to TGF-beta. Thus, we describe a new function for Tax, repression of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.
Asunto(s)
Caveolina 1/metabolismo , Leucemia-Linfoma de Células T del Adulto/metabolismo , Adulto , Caveolina 1/sangre , Caveolina 1/genética , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia-Linfoma de Células T del Adulto/sangre , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Linfocitos T/virología , Activación Transcripcional/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Adult T-cell leukemia (ATL) is a T-cell malignancy etiologically associated with human T-cell leukemia virus type 1 (HTLV-1). Twist, a highly conserved basic helix-loop-helix transcription factor, is a newly identified oncogene. However, there are no reports on Twist expression in ATL. To define the role of Twist in leukemogenesis of ATL, we examined its expression in T-cell lines and PBMC. HTLV-1-infected T-cell lines and ATL cells expressed high levels of Twist compared with uninfected T-cell lines and normal PBMC. Immunohistochemistry showed immunostaining for Twist in ATL cells in ATL lymph nodes and skin lesions. Infection of normal PBMC with HTLV-1 induced Twist expression. Induction of the viral protein Tax in a human T-cell line led to upregulation of Twist. Tax-induced Twist expression involved the NF-kappaB and CREB signaling pathways. Twist augmented Tax-mediated HTLV-1 LTR and NF-kappaB activation. Short interfering RNA against Twist inhibited cell growth of HTLV-1-infected T-cell lines and downregulation of Twist expression in an HTLV-1-infected T-cell line inhibited the expression of Akt1, interleukin-2 receptor alpha chain, and Tax as well as the known target genes of Twist, YB-1 and Akt2. In conclusion, the results suggest that Tax-induced induction of Twist contributes to leukemogenesis of ATL.
RESUMEN
The palindromic sequence motifs (CANNTG) known as E boxes are considered as binding sites for the basic helix-loop-helix (bHLH) class of DNA-binding proteins. Their presence has been reported in the long terminal repeats (LTR) of the HIV-1 and HTLV-1 proviruses. Their close proximity with the TATA region of both LTRs indicates that the bHLH proteins may act as important regulators of the function of proviral transcription. Indeed, observations on HIV-1 and recent results on HTLV-1 underline that these E boxes may be critically involved in the regulation of the proviral transcription of these human retroviruses. Indeed, of the two E boxes flanking the TATA sequences of the HIV-1 provirus, the 3' E box has been implicated in the transcriptional inhibition of viral gene expression. Such a role might also be played by the unique 5' E box present in the HTLV-1 LTR. In both cases, the expression of tissue-specfic bHLH proteins, like TAL1 might counteract the inhibitory effect exerted by E box proteins, thereby increasing proviral transcription. Finally, a phylogenetic study encompassing several subtypes of these two human retroviruses underlines that these E box motifs have recently appeared in the proviral LTRs and may be considered as potential mediators in the establishment of proviral latency.
Asunto(s)
Elementos E-Box/fisiología , Regulación Viral de la Expresión Génica , VIH-1/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Provirus/fisiología , Latencia del Virus , Evolución Molecular , VIH-1/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Modelos Biológicos , Filogenia , Provirus/genética , Secuencias Repetidas Terminales/genética , Transcripción GenéticaRESUMEN
The regulatory Tax protein of HTLV-1 (Human T-cell Leukaemia Virus type 1) is critically involved in the initiation of ATL (adult T-cell leukaemia). Indeed, Tax provides infected T-cells with a growth advantage and with the potential to get transformed through the deregulation of cell-cycle progression and the acquisition of genetic alterations. Considering that leukemias are induced by disturbances in hematopoietic cells development, we hypothesize that the expression of Tax in human immature thymocytes is a prerequisite to the emergence of ATL cells. Studies of alph abeta T-cell development in the thymus have shown that beta-selection, an early important checkpoint, is regulated by transcription factors that are decisive in the control of cell proliferation, differentiation and survival. Interestingly, Tax is endowed with the ability to interfere with the activity of these transcription factors. We therefore propose that the HTLV-1 infection of these specific target thymocytes leads to a transcriptional deregulation of early alphabeta T cell development, thus inducing a pre-leukemogenic event that favours the subsequent proliferation of ATL cells.
Asunto(s)
Transformación Celular Neoplásica , Productos del Gen tax/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Linfocitos T/citología , HumanosRESUMEN
The human T-cell leukemia virus type 1 (HTLV-1) Tax transactivator is known to induce or repress various cellular genes, several of them encoding transcription factors. As Tax is known to deregulate various basic bHLH factors, we looked more specifically at its effect on TAL1 (T-cell acute lymphoblastic leukemia 1), also known as SCL (stem cell leukemia). Indeed, TAL1 is deregulated in a high percentage of T-cell acute lymphoblastic leukemia cells, and its oncogenic properties are well-established. Here we show that Tax induces transcription of this proto-oncogene by stimulating the activity of the TAL1 gene promoter 1b, through both the CREB and NF-kappaB pathways. It was also observed that TAL1 upregulates HTLV-1 promoter activity, in either the presence or the absence of Tax. The viral promoter is inhibited in trans by expression of the E2A protein E47, and TAL1 is able to abrogate this inhibition. These data show the existence of a positive feedback loop between Tax and TAL1 expression and support the notion that this proto-oncogene participates in generation of adult T-cell leukemia/lymphoma by increasing the amount of the Tax oncoprotein but also possibly by its own transforming activities.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación Viral de la Expresión Génica , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Leucemia-Linfoma de Células T del Adulto/virología , Proteínas Proto-Oncogénicas/metabolismo , Sitios de Unión , Línea Celular , Retroalimentación Fisiológica , Células HeLa , Humanos , Modelos Biológicos , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Proteína 1 de la Leucemia Linfocítica T Aguda , Timo/citologíaRESUMEN
BACKGROUND: Activation of telomerase is a critical and late event in tumor progression. Thus, in patients with adult-T cell leukaemia (ATL), an HTLV-1 (Human T cell Leukaemia virus type 1)-associated disease, leukemic cells display a high telomerase activity, mainly through transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). The HBZ (HTLV-1 bZIP) protein coded by the minus strand of HTLV-1 genome and expressed in ATL cells has been shown to increase the transcriptional activity of JunD, an AP-1 protein. The presence of several AP-1 binding sites in the hTERT promoter led us to investigate whether HBZ regulates hTERT gene transcription. RESULTS: Here, we demonstrate using co-transfection assays that HBZ in association with JunD activates the hTERT promoter. Interestingly, the -378/+1 proximal region, which does not contain any AP-1 site was found to be responsible for this activation. Furthermore, an increase of hTERT transcripts was observed in cells co-expressing HBZ and JunD. Chromatin immunoprecipitation (ChIP) assays revealed that HBZ, and JunD coexist in the same DNA-protein complex at the proximal region of hTERT promoter. Finally, we provide evidence that HBZ/JunD heterodimers interact with Sp1 transcription factors and that activation of hTERT transcription by these heterodimers is mediated through GC-rich binding sites for Sp1 present in the proximal sequences of the hTERT promoter. CONCLUSION: These observations establish for the first time that HBZ by intervening in the re-activation of telomerase, may contribute to the development and maintenance of the leukemic process.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Telomerasa/biosíntesis , Transcripción Genética , Regulación hacia Arriba , Proteínas Virales/metabolismo , Inmunoprecipitación de Cromatina , ADN/metabolismo , Dimerización , Humanos , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de los Retroviridae , Factor de Transcripción Sp1/metabolismo , Telomerasa/genéticaRESUMEN
The human pre-T-cell receptor alpha (TCRalpha; pTalpha) gene encodes a polypeptide which associates with the TCRbeta chain and CD3 molecules to form the pre-TCR complex. The surface expression of the pre-TCR is pTalpha dependent, and signaling through this complex triggers an early alphabeta T-cell developmental checkpoint inside the thymus, known as beta-selection. E2A transcription factors, which are involved at multiple stages of T-cell development, regulate the transcription of the pTalpha gene. Here we show that the regulatory protein Tax of the human T-cell leukemia virus type 1 (HTLV-1) efficiently suppresses the E47-mediated activation of the pTalpha promoter. Furthermore, we report that in Tax lentivirally transduced human MOLT-4 T cells, which constitutively express the pTalpha gene, the amount of pTalpha transcripts decreases. Such a decrease is not observed in MOLT-4 cells transduced by a vector encoding the Tax mutant K88A, which is unable to interact with p300. These data underline that Tax inhibits pTalpha transcription by recruiting this coactivator. Finally, we show that the expression of Tax in human immature thymocytes results in a decrease of pTalpha gene transcription but does not modify the level of E47 transcripts. These observations indicate that Tax, by silencing E proteins, down-regulates pTalpha gene transcription during early thymocyte development. They further provide evidence that Tax can interfere with an important checkpoint during T-cell differentiation in the thymus.
Asunto(s)
Regulación hacia Abajo , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano , Glicoproteínas de Membrana/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Timo/inmunología , Diferenciación Celular/genética , Técnicas de Transferencia de Gen , Genes Reporteros , Humanos , Luciferasas/análisis , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Timo/citologíaRESUMEN
The human T-cell leukemia virus HTLV-1 encodes regulatory proteins, Tax, Rex and p30(II), which are involved in the control of viral gene expression at the transcriptional and post-transcriptional levels. Tax localizes in unique nuclear bodies that contain components of the transcription and splicing complexes. In this work, we studied the relative intracellular localizations of Tax, Rex and p30(II). Run-on transcription assays and immunocytochemistry at light and electron microscopy levels indicated that the Tax nuclear bodies included both de novo transcribed RNA and the RNA polymerase II form that is phosphorylated on its carboxy-terminal domain whereas contacts with chromatin were observed at the periphery of these nuclear bodies. Rex first accumulated in nucleolar foci and then spread across the whole nucleus to display a diffuse and punctuate nucleoplasmic distribution. This distribution of Rex was observed in HTLV-1 transformed lymphocytes and in COS cells expressing the HTLV-1 provirus. Rex colocalized with the cellular export factor CRM-1 in the nucleolar foci as well as in the nucleoplasmic foci that did not overlap with Tax nuclear bodies but were found at the boundaries of the Tax bodies. In addition, we demonstrate that p30(II) interacts with Rex and colocalizes with the Rex/CRM-1 complexes in the nucleoli leading to their clearance from the nucleoplasm. Our results suggest that transcripts originating from Tax-induced activation of gene expression at the boundaries of the Tax bodies are transported out of the nucleus by nucleoplasmic Rex/CRM-1 complexes that are first assembled in nucleolar foci. In addition, p30(II) might exert its negative effect on viral RNA transport by preventing the release of the Rex/CRM-1 complexes from sequestration in nucleolar foci. These data support the idea that the transcriptional and post-transcriptional regulation of HTLV-1 gene expression depends on the concentration of select regulatory complexes at specific area of the nucleus.
Asunto(s)
Nucléolo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Regulación Viral de la Expresión Génica/genética , Productos del Gen rex/metabolismo , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Proteínas de los Retroviridae/metabolismo , Animales , Células COS , Línea Celular , Nucléolo Celular/virología , Chlorocebus aethiops , Cricetinae , Humanos , Fosforilación , ARN Polimerasa II/fisiología , ARN Viral/metabolismoRESUMEN
BACKGROUND: The functional state of human telomeres is controlled by telomerase and by a protein complex named shelterin, including the telomeric DNA-binding proteins TRF1, TRF2 and Pot1 involved in telomere capping functions. The expression of hTERT, encoding the catalytic subunit of telomerase, plays a crucial role in the control of lymphocyte proliferation by maintaining telomere homeostasis. It has been previously found that hTERT activity is down-regulated by the human T cell leukaemia virus type 1 (HTLV-1) Tax protein in HTLV-1 transformed T lymphocytes. In this study, we have examined the effects of Tax expression on the transcriptional profile of telomerase and of shelterin in human T lymphocytes. RESULTS: We first provide evidence that the up-regulation of hTERT transcription in activated CD4+ T lymphocytes is associated with a down-regulation of that of TERF1, TERF2 and POT1 genes. Next, the down-regulation of hTERT transcription by Tax in HTLV-1 transformed or in Tax-expressing T lymphocytes is found to correlate with a significant increase of TRF2 and/or Pot1 mRNAs. Finally, ectopic expression of hTERT in one HTLV-1 T cell line induces a marked decrease in the transcription of the POT1 gene. Collectively, these observations predict that the increased transcriptional expression of shelterin genes is minimizing the impact on telomere instability induced by the down-regulation of hTERT by Tax. CONCLUSION: These findings support the notion that Tax, telomerase and shelterin play a critical role in the proliferation of HTLV-1 transformed T lymphocytes.
Asunto(s)
Proteínas de Unión al ADN/genética , Productos del Gen tax/análisis , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Activación de Linfocitos , Linfocitos T/metabolismo , Telomerasa/genética , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Transcripción Genética , Línea Celular , Humanos , Complejo Shelterina , Linfocitos T/virologíaRESUMEN
BACKGROUND: In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1. RESULTS: We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown in C91PL cells increased viral production by these cells. Thus, hnRNP A1 is implicated in the modulation of the level of HTLV-1 gene expression in T cells transformed by this human retrovirus. CONCLUSIONS: These observations provide an insight into a new cellular control of HTLV-1 replication and suggest that hnRNP A1 is likely part of the regulatory mechanisms of the life cycle of this human retrovirus in T cells.