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Antígenos HTLV-I/aislamiento & purificación , Leucemia-Linfoma de Células T del Adulto/terapia , Animales , Descubrimiento de Drogas , Antígenos HTLV-I/metabolismo , Humanos , Japón , Leucemia-Linfoma de Células T del Adulto/etiología , Leucemia-Linfoma de Células T del Adulto/metabolismo , Guías de Práctica Clínica como Asunto , PronósticoRESUMEN
Molecular mimicry between foreign and self Ags is a mechanism of TCR cross-reactivity and is thought to contribute to the development of autoimmunity. The αß TCR A6 recognizes the foreign Ag Tax from the human T cell leukemia virus-1 when presented by the class I MHC HLA-A2. In a possible link with the autoimmune disease human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis, A6 also recognizes a self peptide from the neuronal protein HuD in the context of HLA-A2. We found in our study that the complexes of the HuD and Tax epitopes with HLA-A2 are close but imperfect structural mimics and that in contrast with other recent structures of TCRs with self Ags, A6 engages the HuD Ag with the same traditional binding mode used to engage Tax. Although peptide and MHC conformational changes are needed for recognition of HuD but not Tax and the difference of a single hydroxyl triggers an altered TCR loop conformation, TCR affinity toward HuD is still within the range believed to result in negative selection. Probing further, we found that the HuD-HLA-A2 complex is only weakly stable. Overall, these findings help clarify how molecular mimicry can drive self/nonself cross-reactivity and illustrate how low peptide-MHC stability can permit the survival of T cells expressing self-reactive TCRs that nonetheless bind with a traditional binding mode.
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Presentación de Antígeno/inmunología , Autoantígenos/metabolismo , Secuencia Conservada/inmunología , Proteínas ELAV/metabolismo , Epítopos de Linfocito T/metabolismo , Productos del Gen tax/metabolismo , Imitación Molecular/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Autoantígenos/química , Células Clonales , Reacciones Cruzadas/inmunología , Cristalografía por Rayos X , Proteínas ELAV/química , Proteína 4 Similar a ELAV , Epítopos de Linfocito T/química , Productos del Gen tax/química , Antígeno HLA-A2/biosíntesis , Antígeno HLA-A2/metabolismo , Antígenos HTLV-I/química , Antígenos HTLV-I/metabolismo , Humanos , Neuronas/inmunología , Neuronas/metabolismo , Neuronas/virología , Paraparesia Espástica Tropical/inmunología , Paraparesia Espástica Tropical/metabolismo , Paraparesia Espástica Tropical/virología , Unión Proteica/inmunología , Conformación Proteica , Estabilidad Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
OBJECTIVE: Th1 activation based on a high HTLV-I proviral load is one of the characteristic immunological abnormalities in the peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To clarify the cause of this abnormality with the potential to be one of the therapeutic targets, we analyzed the involvement of interleukin-2 (IL-2)/IL-2 receptor (IL-2R) signaling in HTLV-I and interferon-gamma (IFN-gamma), which is a representative Th1 cytokine, expression in peripheral blood CD4(+) T cells from HAM/TSP patients. PATIENTS AND METHODS: Twelve patients with HAM/TSP were included in the study. After the peripheral blood CD4(+) T cells were treated in cultures under the presence of each anti-IL-2Ralpha, beta,and gamma blocking antiboby for 48 hours, both HTLV-I p19 antigen and IFN-gamma levels in the culture supernatants were measured using ELISA methods. To check the influence on cell proliferation under these culture conditions, the numbers of viable cells were simultaneously determined by MTS assay. RESULTS: Treatment with anti-IL-2Ralpha blocking antibody, but not anti-IL-2Rbeta or anti-IL-2Rgamma blocking antibody, suppressed HTLV-I p19 antigen expression levels. In addition, treatment with all types of anti-IL-2R blocking antibodies also suppressed IFN-gamma expression levels. All of the types of anti-IL-2R blocking antibodies did not inhibit the proliferation. CONCLUSION: These results indicate that IL-2/IL-2R signaling is involved in HTLV-I and IFN-gamma expression on peripheral blood CD4(+) T cells from HAM/TSP patients, suggesting that the interruption of this signaling has therapeutic potential against HAM/TSP in patients with the focus on the down-regulation of Th1 activation based on a high HTLV-I proviral load in the peripheral blood.
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Antígenos HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/inmunología , Interferón gamma/inmunología , Paraparesia Espástica Tropical/inmunología , Paraparesia Espástica Tropical/virología , Receptores de Interleucina-2/inmunología , Adulto , Anciano , Antivirales/farmacología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Femenino , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Humanos , Interferón gamma/efectos de los fármacos , Masculino , Persona de Mediana Edad , Paraparesia Espástica Tropical/sangre , Probabilidad , Receptores de Interleucina-2/efectos de los fármacos , Muestreo , Sensibilidad y Especificidad , Transducción de SeñalRESUMEN
PURPOSE: To examine whether human T-cell leukemia virus type 1 (HTLV-1) could infect a human retinal pigment epithelial (RPE) cell line, ARPE-19, in vitro and to investigate its regulation. METHODS: A coculture system with ARPE-19 and irradiated cells of an HTLV-1-producing T-cell line, MT2 was used to determine the permissivity of RPE to HTLV-1 infection in vitro. The susceptibility to HTLV-1 was assessed by detection of viral DNA using the polymerase chain reaction (PCR), viral mRNA transcripts with reverse transcription PCR (RT-PCR) and viral antigen by immunofluorescence staining. An HTLV-1 Tax-activated HTLV-LTR-luciferase reporter assay was developed to measure viral infection quantitatively. The ICAM-1 expression on cocultured ARPE-19 cells was detected by flow cytometry and an ICAM-1-neutralizing antibody was used to test ICAM-1's role in the HTLV-1 infection of ARPE-19 cells. The regulation of HTLV-1 infection was investigated by culturing ARPE-19 cells with proinflammatory cytokines. RESULTS: HTLV-1 infected ARPE-19 cells in vitro. The infection correlated with elevated expression of intercellular adhesion molecule (ICAM)-1 on the surface of ARPE-19 cells. ICAM-1-neutralizing antibody dramatically inhibited viral infection. Furthermore, proinflammatory cytokines dramatically suppressed HTLV-1 viral infection. CONCLUSIONS: The tropism of HTLV-1 to retinal pigment epithelium could provide an explanation for the pathogenesis of HTLV-1-related ophthalmic diseases. A better understanding of specific roles of proinflammatory cytokines in the development of ophthalmic diseases may be beneficial for treatment.
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Anticuerpos Bloqueadores/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Molécula 1 de Adhesión Intercelular/inmunología , Epitelio Pigmentado Ocular/virología , Línea Celular , Técnicas de Cocultivo , Citocinas/farmacología , ADN Viral/análisis , Citometría de Flujo , Regulación Viral de la Expresión Génica/fisiología , Antígenos HTLV-I/metabolismo , Humanos , Inmunohistoquímica , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/metabolismo , Provirus/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/virología , TransfecciónRESUMEN
The N-terminal domain of the retroviral capsid (CA) protein is one of the least conserved regions encoded in the genome. Surprisingly, the three-dimensional structures of the CA from different genera exhibit alpha-helical structural features that are highly conserved. The N-terminal residues of the human immunodeficiency virus type 1 (HIV-1) and Rous sarcoma virus (RSV) capsid proteins form a beta-hairpin. To determine if this feature is conserved in the retroviral family, we cloned, expressed, purified, and solved the structure of a N-terminal 134 amino acid fragment (CA(134)) from the human T-cell leukemia virus type 1 (HTLV-I) using high resolution nuclear magnetic resonance (NMR) spectroscopy. The CA(134) fragment contains an N-terminal beta-hairpin and a central coiled-coil-like structure composed of six alpha-helices. The N-terminal Pro1 residue contacts Asp54 in the helical cluster through a salt bridge. Thus, the beta-hairpin is conserved and the helical cluster is structurally similar to other retroviral CA domains. However, although the same Asp residue defines the orientation of the hairpin in both the HTLV-1 and HIV-1 CA proteins, the HTLV-I hairpin is oriented away, rather than towards, the helical core. Significant differences were also detected in the spatial orientation and helical content of the long centrally located loop connecting the helices in the core. It has been proposed that the salt bridge allows the formation of a CA-CA interface that is important for the assembly of the conical cores that are characteristic of HIV-1. As HTLV-I forms spherical cores, the salt-bridge feature is apparently not conserved for this function although its role in determining the orientation of the beta-hairpin may be critical, along with the central loop. Comparison of three-dimensional structures is expected to elucidate the relationships between the retroviral capsid protein structure and its function.
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Cápside/química , Virus Linfotrópico T Tipo 1 Humano/química , Secuencia de Aminoácidos , Sitios de Unión , Cápside/metabolismo , Secuencia Conservada , Ciclofilina A/metabolismo , VIH-1/química , Antígenos HTLV-I/química , Antígenos HTLV-I/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Essential HTLV-1 biological functions, like host-cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg147-Leu234] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid-phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non-random conformation of at least 70-80% of the synthetic peptide. Investigation of the 3D-structure of the synthetic peptide will provide useful information for future vaccine and drug-design strategies.
Asunto(s)
Productos del Gen env/química , Péptidos/química , Proteínas Oncogénicas de Retroviridae/química , Acetonitrilos/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Sitios de Unión/inmunología , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Productos del Gen env/inmunología , Productos del Gen env/metabolismo , Antígenos HTLV-I/química , Antígenos HTLV-I/inmunología , Antígenos HTLV-I/metabolismo , Datos de Secuencia Molecular , Concentración Osmolar , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica/inmunología , Proteínas Oncogénicas de Retroviridae/inmunología , Proteínas Oncogénicas de Retroviridae/metabolismo , Propiedades de SuperficieRESUMEN
The p19 matrix (MA) protein of human T-cell leukemia virus type I (HTLV-I) was exposed on the surface of MOLT-4#8 cells in the very early step of the virus infection. Transfer of the virus-binding MOLT-4#8 cells from 4 degrees C to 37 degrees C resulted in increased detection of the viral gp46 and p19 MA protein on the cells, which was, however, inhibited by 4 degrees C or cytochalasin B treatment. These data showed that increased temperature and fluidity of the cell membrane were required for the increased detection of gp46 and p19 after viral adsorption. On the other hand, exposure of the p19 MA protein was not observed on the virus-treated U937 cells although gp46 was detected. This was not due to inefficient binding of the HTLV-I to the U937 cells, since the methanol-fixed cells were p19 MA protein-positive. MOLT-4#8 cells induced marked cell fusion when co-cultured with MT-2 cells, but U937 cells induced no fusion. All of these results indicated that these two cell lines differed in the property of plasma membrane in terms of degradation of HTLV-I envelope after viral adsorption. Uncoating of the HTLV-I might occur on the plasma membrane, especially on MOLT-4#8 cells.
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Membrana Celular/microbiología , Productos del Gen gag/metabolismo , Antígenos HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Adsorción , Línea Celular , Humanos , Fluidez de la Membrana , Temperatura , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The expression of complement receptor 2 (CR2) has been demonstrated in established HTLV-1-transformed cell lines and in 12 studied de novo infected peripheral blood lymphocytes cultures, using 2 HTLV-1 sources. The simultaneous detection of CR2 and HTLV-1 antigens in both co-cultivated and supernatant-infected peripheral blood lymphocytes suggest that the increased CR2 expression is in tandem with the increasing HTLV-1 antigen expression. CR2 up-regulation seen during polyclonal activation is presumably in response to a viral protein, although a cellular factor has not been ruled out. Increasing CR2 expression during early infection suggests its possible involvement in selection or development of subsequent transformation events. Variable levels of CR2 in immortalized cell lines argue against its obligate expression of function in the maintenance of the transformed state. The expression of CR2 in cellular activation of T cells may be stage restricted. This study also expands the cellular distribution for CR2.
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Infecciones por HTLV-I/metabolismo , Receptores de Complemento 3d/metabolismo , Linfocitos T/metabolismo , Línea Celular , Transformación Celular Viral , Antígenos HTLV-I/metabolismo , Humanos , Técnicas In Vitro , Activación de Linfocitos , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Adult T-cell-leukemia (ATL) cells were examined for susceptibility to human T-cell-leukemia virus type I (HTLV-I) tax-specific cytotoxic T lymphocytes (CTL) derived from a patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). These CTL efficiently killed HLA-matched leukemia cells of an ATL patient after overnight incubation. However, ATL cells immediately after isolation from the peripheral blood were only marginally susceptible to the CTL. This is not due to inappropriate expression of major-histocompatibility-complex (MHC)-class-I antigen on the leukemia cells. Addition of synthetic peptide, corresponding to the CTL epitope, to the assay enabled the CTL to kill the fresh ATL cells. Scarcity of HTLV-I antigens in the fresh ATL cells and induction of these antigens by in vitro incubation were demonstrated both on the cell surface and in the cytoplasm. Lectin stimulation augmented synthesis of HTLV-I antigens, but was not essential for the induction. The presence in the culture of human plasma containing a high titer of antibodies to HTLV-I did not affect the induction of HTLV-I expression in the ATL cells. Furthermore, significantly lower levels of HTLV-I tax mRNA were present in the fresh ATL cells than in the cultured ATL cells, whereas the levels of HTLV-I proviral tax gene did not differ among these cells. This suppression of HTLV-I transcription in fresh ATL cells accounts for resistance to the CTL, and could be a reason for the persistence of HTLV-I infection in vivo.
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Productos del Gen tax/inmunología , Antígenos HTLV-I/análisis , Leucemia de Células T/inmunología , Paraparesia Espástica Tropical/inmunología , Linfocitos T Citotóxicos/inmunología , Anciano , Secuencia de Bases , ADN Viral/análisis , Femenino , Antígenos HTLV-I/inmunología , Antígenos HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Leucemia de Células T/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Linfocitos T Citotóxicos/metabolismo , Replicación ViralRESUMEN
Temperature elevation constitutes a beneficial component of the host defence against viral pathogens. However, heat treatment may be detrimental to HTLV-I-infected cells by increasing virion and oncoprotein production. We investigated the effects of thermal elevation on the in vitro replication of HTLV-I (human T-cell leukaemia/lymphoma virus type I) in MT-2 cells, an HTLV-I-transformed lymphoid cell line. We found that HTLV-I replication in MT-2 cells was markedly increased as demonstrated by a nearly 2-fold increase in detection of viral p24 antigen and a 20-fold increase in reverse transcriptase activity during up to 5 h of heat treatment at 42 degrees C. The results suggest that physiologic thermal elevations may induce viral production in HTLV-I-infected individuals.
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Calor , Virus Linfotrópico T Tipo 1 Humano/fisiología , Adulto , Análisis de Varianza , División Celular , Línea Celular Transformada , Supervivencia Celular , Transformación Celular Viral , Preescolar , Femenino , Antígenos HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Cinética , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Replicación ViralRESUMEN
Three protein isoforms are encoded by the human T-cell leukemia/lymphotropic virus type I pX region open reading frames (ORF) I and II through alternative splicing. Both the singly and doubly spliced mRNAs from ORF I encode a single 12-kDa protein (p12I), whereas two distinct proteins of 13 kDa (p13II) and 30 kDa (p30II) are encoded from the ORF II alternatively spliced mRNA. Because the p12I protein is very hydrophobic and poorly immunogenic, we genetically engineered its cDNA by adding a short stretch of amino acids from the highly immunogenic epitope HA1 of influenza virus or the AU1 epitope of bovine papillomavirus. The HA1 epitope was also added to the p13II and p30II proteins, albeit rabbit immune sera raised against synthetic peptides were also available. To determine in which cellular compartments these proteins reside, we transfected the tagged and wild-type cDNAs in HeLa/Tat cells and studied their localization by indirect immunofluorescence. The p12I protein was identified in the cellular endomembranes and, particularly, in the perinuclear area. p13II and p30II were found in the nuclei and nucleoli of the transfected cells, respectively. The presence of the HA1 epitope at the carboxy terminus of p13II and p30II did not interfere with their cellular localization, since the rabbit immune sera demonstrated their presence in the same cellular compartments when the untagged proteins were expressed. The defined localization of these proteins in specific cellular compartments warrants further study of their function.
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Genes pX , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Proteínas de los Retroviridae/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Compartimento Celular , Técnica del Anticuerpo Fluorescente , Antígenos HTLV-I/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Recombinantes/metabolismoRESUMEN
We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.
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Productos del Gen gag/metabolismo , Antígenos HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/ultraestructura , Proteínas Oncogénicas de Retroviridae/metabolismo , Virión/ultraestructura , Anticuerpos Monoclonales , Productos del Gen gag/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Inmunohistoquímica , Proteínas Oncogénicas de Retroviridae/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
A transcriptional trans-acting factor p40tax of human T-cell leukemia virus type I (HTLV-I) functions as an inducer for expression of HTLV-I provirus via activation of the enhancer in the long terminal repeat of HTLV-I. In addition to p40tax and a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, we report here that forskolin, an activator of adenyl cyclase, also induces function of the HTLV-I enhancer. Experiments with mutants of the HTLV-I enhancer revealed that TPA-induced activation was not mediated by solely a 21-base-pair (bp) sequence that is repeated three times in the enhancer, whereas the 21-bp enhancer element can act as a sufficient cis-acting sequence for activation by both p40tax and forskolin. In addition, we found that nuclear factor(s) like the cyclic AMP-responsive element (CRE) binding factor could bind to the HTLV-I 21-bp enhancer element. However, a difference was found in sequences required for activation by p40tax and forskolin. A CRE related sequence present in the 21-bp enhancer element was enough for forskolin-induced activation. On the other hand, p40tax required a much longer sequence that is overlapping but not identical to the CRE related sequence, suggesting that the forskolin-induced cyclic AMP pathway may be partly involved in, but not sufficient for p40tax-mediating trans-activation of the HTLV-I enhancer.
Asunto(s)
Colforsina/farmacología , AMP Cíclico/fisiología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genes Virales , Antígenos HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Factores de Transcripción/metabolismo , Composición de Base , Secuencia de Bases , Cadmio/farmacología , Cloruro de Cadmio , Línea Celular , Núcleo Celular/metabolismo , Elementos de Facilitación Genéticos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Virales/efectos de los fármacos , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Plásmidos , Acetato de Tetradecanoilforbol/farmacología , Transactivadores , TransfecciónRESUMEN
The transactivator protein tax of the human T-cell leukemia virus type I, HTLV-I, is responsible for transactivation of gene expression of viral and cellular genes and is involved in the onset of adult T-cell leukemia, ATL. Genetic deletion studies have implicated a region of the HTLV-I LTR designated as tax-acceptor region, TAR, which is the target of the tax protein. Using antibodies against a tax carboxyterminal synthetic decapeptide the tax protein was purified from an HTLV-I immortalized human T-lymphocyte cell line by immunoaffinity chromatography. The tax protein, purified to apparent homogeneity binds to double-stranded DNA irrespective of its origin from either a nuclear or cytoplasmic fraction of the HTLV-I immortalized cell-line - both of which harbor similar quantities of tax protein. The tax protein binds less to single-stranded DNA and not to single-stranded RNA in vitro. It also binds to DNA-cellulose and heparin-Sepharose. Nuclease treatment of isolated nuclei does not release the tax protein under conditions which release known DNA-binding proteins, such as the myb protein. Transactivation by the tax protein presumably involves host-cell factors, since it does not recognize specific DNA sequences.
Asunto(s)
Antígenos HTLV-I/aislamiento & purificación , Virus Linfotrópico T Tipo 1 Humano/análisis , Factores de Transcripción/aislamiento & purificación , ADN/metabolismo , Antígenos HTLV-I/metabolismo , Transactivadores , Factores de Transcripción/metabolismoRESUMEN
Within the human T-cell leukemia virus type I promoter, there are three copies of a 21-base-pair repeat (hereafter called the tax-responsive element [TRE]) that both contributes to basal promoter activity and mediates induction by the viral activator TAX. We have identified and separated three nuclear proteins that interact with the TRE. The TRE-binding protein designated TREB-3 bound more avidly to a multimerized TRE than to a single-copy TRE, while the other two TRE-binding proteins, TREB-1 and TREB-2, bound equally well to either TRE. TREB-1 has been purified to near homogeneity, and binding activity was localized to a protein of 35 to 43 kilodaltons. The affinity-purified TREB-1 activated transcription from the human T-cell leukemia virus type I promoter in vitro. The purified TREB-1 fraction contained activating transcription factor binding activity and showed a cooperative interaction with the TATA-binding factor (TFIID) on the adenovirus E4 promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Antígenos HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular Transformada , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Antígenos HTLV-I/aislamiento & purificación , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Transactivadores , Factores de Transcripción/aislamiento & purificaciónRESUMEN
The HTLV-I antigen (Ag) was detected in short-term cultured peripheral blood mononuclear cells (PBMNC) from 44% of patients with HTLV-I-associated myelopathy (HAM), using the indirect immunofluorescence method. The HTLV-I Ag-positive cells accounted for less than 4% of cultured PBMNC in all but one case. The kinetics and mechanism of HTLV-I Ag expression in cultured cells were these studied in this individual with about 20% positive cells. HTLV-I Ag was 0.3% at 6 h after the culture and the number of positive cells increased to 9.3% at 48 h. The sera from HAM patients had a suppressive effect on the expression of HTLV-I Ag in the cultured cells. This suppression was more potent in sera from patients with a high than with low antibody titer. There were no correlations between the HTLV-I Ag expression in cultured cells and the various clinical and laboratory findings, in each case.
Asunto(s)
Antígenos HTLV-I/metabolismo , Infecciones por HTLV-I/inmunología , Leucocitos Mononucleares/inmunología , Adulto , Células Cultivadas , Femenino , Humanos , Idoxuridina/farmacología , Técnicas In Vitro , Leucocitos Mononucleares/citología , Masculino , Persona de Mediana EdadRESUMEN
To understand the mechanisms of oncogenesis by human T-cell leukemia virus type I, we have investigated the ability of the tax1, protein to modulate transcription of protooncogenes. By using a transient cotransfection assay, we report that the protooncogene fos promoter is transactivated by tax1 in a variety of cell types. Two regions containing upstream sequences between positions -362/-324 and -323/-276 of the c-fos promoter responded to this activation and also conferred tax1 responsiveness to the heterologous herpesvirus thymidine kinase promoter. These two sequences include elements mediating the induction by v-sis-conditioned medium and serum, phorbol ester, or epidermal growth factor, respectively. Furthermore, expression of the endogenous c-fos gene was activated by tax1 in human T-cell leukemia virus type I-infected cell lines. In contrast, no trans-activation of the c-myc or c-Ha-ras promoter was observed.