RESUMEN
Despite the utility of cyclooxygenase (COX) inhibition as an antiinflammatory strategy, prostaglandin (PG) products of COX-1 and -2 provide important regulatory functions in some pathophysiological states. Scattered reports suggest that COX inhibition may also promote adverse drug events. Here we demonstrate a protective role for endogenous COX-derived products in a murine model of acetaminophen (APAP)-induced acute liver injury. A single hepatotoxic dose caused the selective induction of COX-2 mRNA and increased PGD2 and PGE2 levels within the livers of COX(+/+) male mice suggesting a role for COX-2 in this model of liver injury. APAP-induced hepatotoxicity and lethality were markedly greater in COX-2(-/-) and (-/+) mice in which normal PG responsiveness is altered. The significantly increased toxicity linked to COX-2 deficiency could be mimicked using the selective COX-2 inhibitory drug, celecoxib, in COX(+/+) mice and was not due to alterations in drug-protein adduct formation, a surrogate for bioactivation and toxicity. Microarray analyses indicated that increased injury associated with COX-2 deficiency coincided, most notably, with a profoundly impaired induction of heat shock proteins in COX-2(-/+) mice suggesting that PGs may act as critical endogenous stress signals following drug insult. These findings suggest that COX-2-derived mediators serve an important hepato-protective function and that COX inhibition may contribute to the risk of drug-induced liver injury, possibly through both nonimmunological and immunological pathways.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Acetaminofén/toxicidad , Animales , Celecoxib , Enfermedad Hepática Inducida por Sustancias y Drogas/mortalidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Cartilla de ADN/química , Dinoprostona/biosíntesis , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Immunoblotting , Isoenzimas/antagonistas & inhibidores , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Prostaglandina D2/biosíntesis , Pirazoles , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , Tasa de SupervivenciaRESUMEN
Mammalian, plant, and Schizosaccharomyces pombe eukaryotic initiation factor-3 (eIF3) contains a protein homologous to the product of int-6 (eIF3e), a frequent integration site of mouse mammary tumor viruses. By contrast, Saccharomyces cerevisiae does not encode a protein closely related to eIF3e/Int-6. Here, we characterize a novel S. cerevisiae protein (Pci8p, Yil071cp) that contains a PCI (proteasome-COP9 signalosome-eIF3) domain conserved in eIF3e/Int-6. We show that both Pci8p and human eIF3e/Int-6 expressed in budding yeast interact with the yeast eIF3 complex in vivo and in vitro by binding to a discrete segment of its eIF3b subunit Prt1p and that human eIF3e/Int-6 interacts with the human eIF3b segment homologous to the Pci8p-binding site of yeast Prt1p. These results refine our understanding of subunit interactions in the eIF3 complex and suggest structural similarity between human eIF3e/Int-6 and yeast Pci8p. However, deletion of PCI8 had no discernible effect on cell growth or translation initiation as judged by polysome analysis, suggesting that Pci8p is not required for the essential function of eIF3 in translation initiation. Motivated by the involvement of Int-6 in transcriptional control, we investigated the effects of deleting PCI8 on the total mRNA expression profile by oligonucleotide microarray analysis and found reduced mRNA levels for a subset of heat shock proteins in the pci8Delta mutant. We discuss possible dual functions of Pci8p and Int-6 in transcriptional and translational control.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Proteínas Fúngicas/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Saccharomyces cerevisiae/química , Sitios de Unión , Complejo del Señalosoma COP9 , Perfilación de la Expresión Génica , Humanos , Complejos Multiproteicos , Péptido Hidrolasas , Factor 3 Procariótico de Iniciación , Subunidades de Proteína , Proteínas/fisiología , ARN Mensajero/análisis , Transcripción GenéticaRESUMEN
Drug-induced hepatotoxicity causes significant morbidity and mortality and is a major concern in drug development. This is due, in large part, to insufficient knowledge of the mechanism(s) of drug-induced liver injury. In order to address this problem, we have evaluated the modulation of gene expression within the livers of mice treated with a hepatotoxic dose of acetaminophen (APAP) using high-density oligonucleotide microarrays capable of determining the expression profile of >11,000 genes and expressed sequence tags (ESTs). Significant alterations in gene expression, both positive and negative, were noted within the livers of APAP-treated mice. APAP-induced toxicity affected numerous aspects of liver physiology causing, for instance, >twofold increased expression of genes that encode for growth arrest and cell cycle regulatory proteins, stress-induced proteins, the transcription factor LRG-21, suppressor of cytokine signaling (SOCS)-2-protein, and plasminogen activator inhibitor-1 (PAI-1). A number of these and other genes and ESTs were detectable within the liver only after APAP treatment suggesting their potential importance in propagating or preventing further toxicity. These data provide new directions for mechanistic studies that may lead to a better understanding of the molecular basis of drug-induced liver injury and, ultimately, to a more rational design of safer drugs.
Asunto(s)
Acetaminofén/toxicidad , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Secuencia de Bases , Cartilla de ADN , Hígado/metabolismo , Ratones , Datos de Secuencia MolecularRESUMEN
p53 plays a pivotal role in transmitting signals from many forms of genotoxic stress to genes and factors that control the cell cycle and apoptosis. We have previously shown that the human T-lymphotropic virus type I Tax protein can inhibit p53 function. Recently we reported that Tax inhibits p53 function in Jurkat cells and mouse embryo fibroblasts through a mechanism involving the nuclear factor kappa B pathway and correlates with phosphorylation on serines 15 and 392 of p53. However, several groups have also observed a mechanism that correlates with p300 binding of Tax. To address this controversy and to determine the mechanism by which Tax inhibits p53 function, we examined the activation functions of Tax required for p53 inhibition. In HeLa and H1299 cells the cAMP-response element-binding protein/activating transcription factor activation function is essential, as demonstrated by the Tax mutants M47 and K88A. In addition, expression of exogenous p300 in H1299 cells allows full recovery of p53 transactivation in the presence of Tax. Consistent with p300 being a limiting factor in H1299, Saos-2, and HeLa cells, we found that the level of endogenous p300 is relatively low in these cells compared with Jurkat cells or the human T-lymphotropic virus type I-infected C81 and MT2 cells. Thus our data suggests that Tax utilizes distinct mechanisms to inhibit p53 function that are cell type-dependent.
Asunto(s)
Proteínas de Unión al ADN , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Transducción de Señal , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Factor de Transcripción Activador 1 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Productos del Gen tax/genética , Genes Reporteros , Histona Acetiltransferasas , Humanos , Linfocitos/metabolismo , Linfocitos/virología , FN-kappa B/metabolismo , Especificidad de Órganos , Fosforilación , Factores de Transcripción/metabolismo , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBPRESUMEN
The p53 protein plays a pivotal role in transmitting signals from many forms of genotoxic stress to genes and factors that control aspects of the cell cycle and death. Although mutated in approximately 60% of all human cancers, only a minority of human T-lymphotropic virus type 1 (HTLV-1)-transformed cells carry p53 mutations. Nevertheless, the p53 protein in HTLV-1-transformed cells is functionally inactive. We have previously demonstrated that the HTLV-1 Tax protein can inhibit p53 trans-activation function. Tax does not accomplish this by directly binding to p53, but rather by a unique mechanism that includes constitutive phosphorylation of p53 at Ser-15 and Ser-392. Analysis of Tax mutants in lymphocytes demonstrates that Tax-induced p53 inhibition correlates with the ability of Tax to activate NF-kappaB, but not p300 binding or CREB trans-activation. Consistent with these results, expression of the I-kappaBalpha(S32,36A) mutant that blocks NF-kappaB activation blocks Tax-mediated p53 inhibition. We further demonstrate the importance of Tax activation of NF-kappaB in p53 inhibition, using p65 knockout (KO) mouse embryo fibroblasts (MEFs). In the absence of p65 Tax could not inhibit p53. Tax does activate IKKbeta in the p65 KO MEFs, indicating that prenuclear events of NF-kappaB activation are not sufficient for Tax-mediated p53 inhibition, but rather NF-kappaB transcriptional activation is critical. Importantly, using phosphospecific antibodies, we demonstrate that phosphorylation of p53 at Ser-15 and Ser-392 correlates with Tax-mediated inhibition. In addition, mutation of p53 at Ser-15 and Ser-392 to alanines renders p53 resistant to Tax inhibition. This report reviews p53 inhibition by Tax and presents our current model.
Asunto(s)
Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Linfocitos/virología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Transformada , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Ratones , Activación Transcripcional , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Human T cell lymphotropic virus type II (HTLV-2) was originally isolated from a patient with a hairy T cell leukemia. It has been associated with rare cases of CD8(+) T lymphoproliferative disorders, and has a controversial role as a pathogen. The loss of p53 function, as a consequence of mutation or inactivation, increases the chances of genetic damage. Indeed, the importance of p53 as a tumor suppressor is evident from the fact that over 60% of all human cancers have a mutant or inactive p53. p53 status has been extensively studied in HTLV-1-infected cell lines. Interestingly, despite the fact that p53 mutations have been found in only a minority of cells, the p53 functions were found to be impaired. We have analyzed the functional activity of the p53 tumor suppressor in cells transformed with HTLV-2 subtypes A and B. As with HTLV-1-infected cells, abundant levels of the p53 protein are detected in HTLV-2 virus-infected cell lines. Using p53 reporter plasmid or induction of p53-responsive genes in response to gamma-irradiation, the p53 was found to be transcriptionally inhibited in HTLV-2-infected cells. Interestingly, although Tax-2A and-2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B in T cells, but not in fibroblasts.
Asunto(s)
Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/fisiología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Transformada , Infecciones por HTLV-I/virología , Infecciones por HTLV-II/virología , Humanos , Ratones , Linfocitos T/virología , Transcripción Genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We have analyzed the functional activity of the p53 tumor suppressor in human T-cell lymphotropic virus type 2 (HTLV-2)-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, both in transient-transfection assays using a p53 reporter plasmid and in induction of p53-responsive genes in response to gamma irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. In transient-cotransfection assays, Tax-1 and Tax-2B inactivated p53 by 80%, while Tax2A reduced p53 activity by 20%. In addition, Tax-2A does not increase the steady-state level of cellular p53 as well as Tax-1 or -2B does in the same assays. Cotransfection assays demonstrated that Tax-2A could efficiently transactivate CREB-responsive promoters to the same level as Tax-1 and Tax-2B, indicating that the protein was functional. This report provides evidence of the first functional difference between the HTLV-2A and -2B subtypes. This comparison of the action of HTLV-1 and HTLV-2 Tax proteins on p53 function will provide important insights into the mechanism of HTLV transformation.
Asunto(s)
Transformación Celular Viral , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Transformada , Rayos gamma , Regulación de la Expresión Génica , Productos del Gen tax/genética , Infecciones por HTLV-II/virología , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Humanos , Células Jurkat , Fosforilación , Linfocitos T/virología , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/efectos de la radiaciónRESUMEN
p53 plays a key role in guarding cells against DNA damage and transformation. We previously demonstrated that the human T-cell lymphotropic virus type 1 (HTLV-1) Tax can inactivate p53 transactivation function in lymphocytes. The present study demonstrates that in T cells, Tax-induced p53 inactivation is dependent upon NF-kappaB activation. Analysis of Tax mutants demonstrated that Tax inactivation of p53 function correlates with the ability of Tax to induce NF-kappaB but not p300 binding or CREB transactivation. The Tax-induced p53 inactivation can be overcome by overexpression of a dominant IkappaB mutant. Tax-NF-kappaB-induced p53 inactivation is not due to p300 squelching, since overexpression of p300 does not recover p53 activity in the presence of Tax. Further, using wild-type and p65 knockout mouse embryo fibroblasts (MEFs), we demonstrate that the p65 subunit of NF-kappaB is critical for Tax-induced p53 inactivation. While Tax can inactivate endogenous p53 function in wild-type MEFs, it fails to inactivate p53 function in p65 knockout MEFs. Importantly, Tax-induced p53 inactivation can be restored by expression of p65 in the knockout MEFs. Finally, we present evidence that phosphorylation of serines 15 and 392 correlates with inactivation of p53 by Tax in T cells. This study provides evidence that the divergent NF-kappaB proliferative and p53 cell cycle arrest pathways may be cross-regulated at several levels, including posttranslational modification of p53.
Asunto(s)
Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Proteína p300 Asociada a E1A , Fibroblastos/citología , Fibroblastos/metabolismo , Productos del Gen tax/genética , Humanos , Proteínas I-kappa B/genética , Células Jurkat , Ratones , Ratones Noqueados , Mutación , Proteínas Nucleares/metabolismo , Fosforilación , Linfocitos T/citología , Linfocitos T/metabolismo , Transactivadores/metabolismo , Factor de Transcripción ReIARESUMEN
Cyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G(2) phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G(1). This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G(2) and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1, vpr, which arrests infected cells at the G(2)/M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions.
Asunto(s)
Ciclo Celular , Regulación Viral de la Expresión Génica , Productos del Gen tat/metabolismo , VIH-1/genética , Extractos Celulares , Epítopos/genética , Fase G1 , Fase G2 , Productos del Gen tat/genética , Productos del Gen vpr/genética , Productos del Gen vpr/metabolismo , Duplicado del Terminal Largo de VIH , Células HeLa , Hemaglutininas/genética , Humanos , Factor de Transcripción Sp1/metabolismo , Transcripción Genética , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation. PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity. In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein. In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP. PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold. Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding. Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain. Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF. In contrast, p300 stimulation of Tax transactivation is HAT dependent. These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Regulación Viral de la Expresión Génica , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Proteínas de Saccharomyces cerevisiae , Activación Transcripcional , Sitios de Unión , Proteína de Unión a CREB , ADN Viral/metabolismo , Productos del Gen tax/genética , Histona Acetiltransferasas , Humanos , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta , Secuencias Repetidas Terminales , Transactivadores/metabolismo , Factores de Transcripción , Transcripción Genética , Factores de Transcripción p300-CBPRESUMEN
Inhibition of p53 function, through either mutation or interaction with viral or cellular transforming proteins, correlates strongly with the oncogenic potential. Only a small percentage of human T-cell lymphotropic virus type 1 (HTLV-1)-transformed cells carry p53 mutations, and mutated p53 genes have been found in only one-fourth of adult T-cell leukemia cases. In previous studies, we demonstrated that wild-type p53 is stabilized and transcriptionally inactive in HTLV-1-transformed cells. Further, the viral transcriptional activator Tax plays a role in both the stabilization and inactivation of p53 through a mechanism involving the first 52 amino acids of p53. Here we show for the first time that phosphorylation of p53 inactivates p53 by blocking its interaction with basal transcription factors. Using two-dimensional peptide mapping, we demonstrate that peptides corresponding to amino acids 1 to 19 and 387 to 393 are hyperphosphorylated in HTLV-1-transformed cells. Moreover, using antibodies specific for phosphorylated Ser15 and Ser392, we demonstrate increased phosphorylation of these amino acids. Since HTLV-1 p53 binds DNA in a sequence-specific manner but fails to interact with TFIID, we tested whether phosphorylation of the N terminus of p53 affected p53-TFIID interaction. Using biotinylated peptides, we show that phosphorylation of Ser15 alone inhibits p53-TFIID interaction. In contrast, phosphorylation at Ser15 and -37 restores TFIID binding and blocks MDM2 binding. Our studies provide evidence that HTLV-1 utilizes the posttranslational modification of p53 in vivo to inactivate function of the tumor suppressor protein.
Asunto(s)
Transformación Celular Viral , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteínas Nucleares , Proteína p53 Supresora de Tumor/metabolismo , Sitios de Unión , Línea Celular Transformada , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Fosforilación , Conformación Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Serina/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción TFII/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/químicaRESUMEN
Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia. HTLV-1 transforms lymphocytes, and there is increasing evidence that the virus-encoded protein, Tax, plays a primary role in viral transformation. We have shown that wild-type p53 in HTLV-1-transformed cells is stabilized. This study was initiated to directly analyze whether the p53 in HTLV-1-transformed cell lines was transcriptionally active and to identify the viral gene product responsible for stabilization and inactivation. Transfection experiments using a p53-responsive reporter plasmid and gamma-irradiation studies demonstrate that the wild-type p53 in HTLV-1-transformed cell lines is not fully active. Further, we demonstrate that the HTLV-1-transforming protein, Tax, stabilizes and inactivates p53 function. Cotransfection of Tax with p53 results in a greater than 10-fold reduction in p53 transcription activity. Using Ga14-p53 fusion proteins, we demonstrate that Tax inhibition of p53 transactivation function is independent of sequence-specific DNA binding. Moreover, Tax inhibits p53 function by interfering with the activity of the N-terminal activation domain (amino acids 1 to 52). We conclude that Tax is involved in the inactivation of p53 function and stabilization of p53 in HTLV-1-infected cells. The functional interference of p53 function by Tax may be important for transformation and leukemogenesis.
Asunto(s)
Transformación Celular Viral/genética , Regulación Viral de la Expresión Génica , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Proteína p53 Supresora de Tumor/genética , Línea Celular Transformada , Humanos , Activación TranscripcionalRESUMEN
DESIGN: Infection of the human promyelocytic cell line HL-60 with NL4-3, a molecularly cloned HIV-1 strain that productively infects T cells, results in adaptation of the virus and production of a variant, NL4-3(M). Unlike NL4-3, NL4-3(M) has a rapid cytopathic effect in HL-60 and other myeloid cell lines. OBJECTIVE: To demonstrate that the tropism of NL4-3(M) is extended to primary monocyte-derived macrophages (MDM), and to determine whether the envelope gene, env, of NL4-3(M) is responsible for cytopathicity in HL-60 cells and replication in MDM. METHODS: A chimeric virus (NL4-3envA) containing the majority of env of NL4-3(M) was generated, and tested for virus replication and cytopathic effect in H9 and HL-60 cells, as well as for virus replication in primary MDM. To assess virus replication, the cultures were analyzed for expression of viral envelope glycoproteins on the infected cells and production of extracellular HIV-1 p24 antigen. Cytopathic effect on HL-60 cells was evaluated by monitoring the viabilities of the cultures. In addition, the majority of env of NL4-3envA was sequenced. RESULTS: The biological phenotypes of NL4-3, NL4-3(M), and NL4-3envA are distinctly different. Although both NL4-3(M) and NL4-3envA replicate in MDM, only NL4-3(M) is rapidly cytopathic in HL-60 cells. Nine amino-acid changes were identified within the envelope glycoproteins of NL4-3envA compared with NL4-3. CONCLUSIONS: The viral determinants of NL4-3(M) sufficient to extend the tropism of this virus to MDM reside, in part, in env. These genetic determinants are distinct from the viral determinants that control the cytopathic phenotype of this virus in HL-60 cells.
Asunto(s)
VIH-1/fisiología , Macrófagos/virología , Sitios de Unión , Efecto Citopatogénico Viral , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , Células HL-60 , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Fenotipo , Virus Reordenados/fisiologíaRESUMEN
Tax1, a potent activator of human T-cell lymphotropic virus type 1 (HTLV-1) transcription, has been shown to modulate expression of many cellular genes. Tax1 does not bind DNA directly but regulates transcription through protein-protein interactions with sequence-specific transcription factors. Using the yeast two-hybrid system to screen for proteins which interact with Tax1, we isolated the B subunit of the CCAAT binding protein NF-Y from a HeLa cDNA library. The interaction of Tax1 with NF-YB was specific in that NF-YB did not interact with a variety of other transcription factors, including human immunodeficiency virus Tat, human papillomavirus E6, and Bicoid, or with the M7 (amino acids 29CP-AS) Tax1 mutant. However, NF-YB did interact with the C-terminal Tax1 mutants M22 (130TL-AS) and M47 (319LL-RS). We also show that in vitro-translated NF-YB specifically bound to a glutathione S-transferase-Tax1 fusion protein. Further, Tax1 coimmunoprecipitated with NF-Y from nuclear extracts of HTLV-1-transformed cells, providing evidence for in vivo interaction of Tax1 and NF-YB. We further demonstrate that Tax1 specifically activated the NF-Y-responsive DQbeta promoter, as well as a minimal promoter which contains only the Y-box element. In addition, mutation of the Y-box element alone abrogated Tax1-mediated activation. Taken together, these data indicate that Tax1 interacts with NF-Y through the B subunit and that this interaction results in activation of the major histocompatibility complex class II promoter. Through activation of this and other NF-Y driven promoters, the Tax1-NF-Y interaction may play a critical role in causing cellular transformation and HTLV-1 pathogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Productos del Gen tax/metabolismo , Genes MHC Clase II/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Factores de Transcripción/metabolismo , Proteínas Potenciadoras de Unión a CCAAT , Extractos Celulares , Línea Celular Transformada , Núcleo Celular , Clonación Molecular , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica/fisiología , Productos del Gen tax/genética , Antígenos HLA-DQ/genética , Cadenas beta de HLA-DQ , Células HeLa , Humanos , Mutación , Pruebas de Precipitina , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T , Factores de Transcripción/genéticaRESUMEN
We have previously shown that the parathyroid hormone-related protein (PTHrP) promoter contains binding sites for transcription factors Ets1 and Sp1 and that human T-cell lymphotropic virus type I (HTLV-I) Tax cooperates with Ets1 to transactivate the PTHrP P2 promoter. Using the yeast two-hybrid interaction system, we now provide evidence that Tax interacts with Ets1. Moreover, a double mutation (D22A,C23S) in the Tax protein that abrogated the Tax/Ets1 interaction also inhibited the Tax/Ets1 cooperative effect, suggesting that the interaction between Tax and Ets1 is important for transactivation of the PTHrP promoter. In coimmunoprecipitation assays, we find that Tax facilitates the interaction between Ets1 and Sp1, forming a ternary complex. When the Sp1 site in the PTHrP promoter was mutated, the Tax/Ets1 cooperative effect was dramatically decreased. This suggests that Sp1 plays an important role in the Ets1-dependent Tax transactivation of the PTHrP P2 promoter. Finally, we demonstrate that Gal4-Tax is a strong activator of the Gal PTHrP promoter, implying that Tax contributes directly to the transcriptional activation of the promoter. We propose a model in which the Tax/Ets1 cooperative effect on the PTHrP P2 promoter is based on the ability of Tax, Ets1, and Sp1 to form a ternary complex on the template DNA. Tax facilitates the interaction of Ets1/Sp1 and participates directly in the transcription initiation process.
Asunto(s)
Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Factor de Transcripción Sp1/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Mutación , Proteína Relacionada con la Hormona Paratiroidea , Regiones Promotoras Genéticas/genética , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas c-ets , Activación TranscripcionalRESUMEN
To understand how different cell types might influence the generation of viral variants, we have examined the differences in the viral life cycle of the HIV-1 isolate, NL4-3, in the human promyelocytic cell line, HL-60, and the human T cell line, H9. NL4-3 harvested from H9 cells productively infected and was cytopathic to H9 and HL-60 cells. However, the cytopathic effect was delayed in HL-60 cells compared to that seen in H9 cells, suggesting that NL4-3 replication was restricted in myeloid cells. This restriction was overcome by production of a variant virus, NL4-3 (M), which replicated efficiently in HL-60 cells. Measurements of the kinetics of entry of NL4-3 in H9 and HL-60 cells and NL4-3 (M) in HL-60 cells demonstrated that the timing of viral entry into each cell line was similar. However, quantitation of the amount of newly reverse-transcribed NL4-3 DNA in H9 and HL-60 cells revealed that NL4-3-infected H9 cells and NL4-3 (M)-infected HL-60 cells contained consistently more newly reverse-transcribed DNA than NL4-3-infected HL-60 cells. This difference was further amplified by inefficient spread of the virus throughout the HL-60 culture. Together these results suggest that the efficiency of NL4-3 infection of HL-60 cells is restricted at early steps in the viral life cycle and may be restricted at late steps as well.