RESUMEN
As coxsackievirus B3 (CoxB3) and adenoviruses may cause acute myocarditis and inflammatory cardiomyopathy, isolation of the common coxsackievirus-adenovirus-receptor (CAR) has provided an interesting new target for molecular antiviral therapy. Whereas many viruses show high mutation rates enabling them to develop escape mutants, mutations of their cellular virus receptors are far less likely. We report on antiviral efficacies of CAR gene silencing by short hairpin (sh)RNAs in the cardiac-derived HL-1 cell line and in primary neonatal rat cardiomyocytes (PNCMs). Treatment with shRNA vectors mediating RNA interference against the CAR resulted in almost complete silencing of receptor expression both in HL-1 cells and PNCMs. Whereas CAR was silenced in HL-1 cells as early as 24 h after vector treatment, its downregulation in PNCMs did not become significant before day 6. CAR knockout resulted in inhibition of CoxB3 infections by up to 97% in HL-1 cells and up to 90% in PNCMs. Adenovirus was inhibited by only 75% in HL-1 cells, but up to 92% in PNCMs. We conclude that CAR knockout by shRNA vectors is efficient against CoxB3 and adenovirus in primary cardiac cells, but the efficacy of this approach in vivo may be influenced by cell type-specific silencing kinetics in different tissues.
Asunto(s)
Infecciones por Adenoviridae/terapia , Infecciones por Coxsackievirus/terapia , Terapia Genética/métodos , Miocarditis/terapia , Interferencia de ARN , Receptores Virales/genética , Adenoviridae , Animales , Línea Celular , Células Cultivadas , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Enterovirus Humano B , Silenciador del Gen , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Miocarditis/virología , Miocitos Cardíacos/virología , ARN Interferente Pequeño/administración & dosificación , Ratas , Replicación Viral/genéticaRESUMEN
The C-terminal binding protein (CtBP) acts as a transcriptional corepressor upon recruitment to transcriptional regulators. In contrast, interaction between CtBP and the adenovirus E1A protein is required for efficient activation of E1A-responsive genes, suggesting that E1A might block CtBP-mediated repression. Recruitment of CtBP to a promoter, either as a Gal4CtBP fusion or through an interaction with a Gal4 fusion protein expressing the CtBP interacting domain (CID) of E1A, resulted in transcriptional repression. The second exon of E1A, containing the CID, alleviated repression by Gal4E1ACID-recruited CtBP, but not Gal4CtBP-mediated repression, suggesting that E1A prevented repression by blocking promoter recruitment of CtBP. E1ACID was also sufficient to derepress transcription from several cotransfected promoter constructs. Furthermore, inducible expression of E1ACID in established cell lines resulted in significant changes of endogenous gene expression, possibly by sequestration of CtBP. Together, these data indicated that CtBP might act as a wide-range regulator of transcription. Although CtBP was shown to interact with histone deacetylases (HDACs), transcriptional repression by a Gal4CtBP fusion protein was not sensitive to inhibition of HDACs by trichostatin A (TSA). In contrast, TSA eliminated E1ACID derepression of E1A second exon-responsive promoters. Although the reason for this difference remains to be experimentally verified, it is possible that the requirement for HDACs might differ depending on the mechanism by which CtBP becomes promoter recruited.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Fosfoproteínas/metabolismo , Proteínas Represoras/metabolismo , Proteínas E1A de Adenovirus/genética , Oxidorreductasas de Alcohol , Sitios de Unión/genética , Línea Celular , Exones , Genes Reporteros , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Transcripción GenéticaRESUMEN
Binding of the C-terminal binding protein, CtBP, to the adenovirus E1A moiety of a Gal4-E1A fusion protein abolishes conserved region (CR) 1-dependent transcription activation. In contrast, a non-promoter targeted E1A peptide, capable of binding CtBP, can induce transcription from the proliferating cell nuclear antigen (PCNA) promoter. CtBP is shown here to bind the histone deacetylase HDAC1, suggesting that a promoter targeted CtBP-HDAC1 complex can silence transcription from the PCNA promoter through a deacetylation mechanism. Expression of the CtBP binding domain of E1A is sufficient to alleviate repression, possibly due to the displacement of the CtBP-HDAC1 complex from the promoter.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Desacetilasas/metabolismo , Fosfoproteínas/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Oxidorreductasas de Alcohol , Sitios de Unión , Proteínas de Unión al ADN/genética , Exones , Histona Desacetilasa 1 , Histona Desacetilasas/genética , Humanos , Fosfoproteínas/genética , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales CultivadasRESUMEN
Ras-transformed cells often show high levels of expression of activating protein-1 and Ets and of genes regulated by these transcription factors. In analogy with the effects of transient stimulation of Ras, it is assumed that the increase in transcription-factor transactivation in stably transformed cells is due to Ras-induced constitutive activation of mitogen-activated protein kinases. However, this has not been extensively studied. Using specific substrate peptides, we have examined here the activities of two types of mitogen-activated protein kinase, extracellular-signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK), in [Val12]Ras-transformed rat embryo fibroblast cell lines. These activities were elevated 2-3-fold in Ras-transformed cells compared with non-transformed cells with a similar growth rate. Increased ERK activity was not necessarily accompanied by a similar increase in JNK activity. In transformed cells, ERK and JNK activities could be stimulated fourfold and ninefold by phorbol ester and ultraviolet-light treatment, respectively, indicating that only a fraction of these enzymes were constitutively activated in these cells. It has been suggested that inactive JNK downregulates c-Jun transcriptional activity by binding to the c-Jun delta-domain. No decrease in delta-inhibitor activity could be demonstrated in Ras-transformed cells compared with control cells, consistent with the presence of mainly inactive JNK in transformed cells. Treatment of transformed cells wih benzodiazepine 5B, an inhibitor of Ras farnesylation, decreased ERK and JNK activities, and concomitantly caused morphological reversion, reduced growth rate, and normalization of transformation-related gene expression. We conclude that in stably Ras-transformed cells the moderately increased ERK/JNK activities are not coregulated, and that ERK rather than JNK activity correlated with transformation-related gene expression.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Transformación Celular Neoplásica , Genes ras , Proteínas Quinasas Activadas por Mitógenos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal/fisiología , Animales , Benzodiazepinas/farmacología , Northern Blotting , Western Blotting , Línea Celular Transformada , Histonas/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteína Básica de Mielina/metabolismo , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/genética , ARN Mensajero/metabolismo , Ratas , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/genética , Factores de Transcripción/farmacología , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The adenovirus E1A-243R protein has the ability to force a resting cell into uncontrolled proliferation by modulating the activity of key targets in cell cycle control. Most of these regulatory mechanisms are dependent on activities mapping to conserved region 1 (CR1) and the non-conserved N-terminal region of E1A. We have previously shown that CR1 functions as a very patent transactivator when it is tethered to a promoter through a heterologous DNA binding domain. However, artificial DNA binding was not sufficient to convert full-length E1A-243R to a transactivator. Thus, an additional function(s) of the E1A-243R protein modulates the effect of CR1 in transcription regulation. Here we demonstrate that a 44 amino acid region at the extreme C-terminus of ElA inhibited transactivation by a Gal4-CR1 fusion protein. Inhibition correlated with binding of the nuclear 48 kDa C-terminal binding protein (CtBP), which has been implicated in E1A-mediated suppression of the metastazing potential of tumour cells. This might suggest that CtBP binding can regulate E1A-mediated transformation by modulating CR1-dependent control of transcription.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Proteínas de Unión al ADN/metabolismo , Fosfoproteínas/metabolismo , Transcripción Genética , Activación Transcripcional , Oxidorreductasas de Alcohol , Sitios de Unión , Transformación Celular Neoplásica , Células HeLa , Humanos , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Relación Estructura-ActividadRESUMEN
Different Gal4 fusion proteins, expressing unrelated transcription activator domains, were found to activate transcription from promoters containing dimerized AP1 DNA binding sites. Transactivation was dependent on the first 74 amino acids of Gal4. A direct interaction between Gal4 and c-Jun was demonstrated using a GSTGal4 fusion protein and in vitro translated human c-Jun. The interaction required the zinc finger containing DNA binding domain of Gal4 and the basic-leucine zipper region of c-Jun. These results demonstrated that the specificity of Gal4 fusion proteins in transient transfection experiments in mammalian cells is not restricted to reporters containing Gal4 binding sites, but also includes promoters containing AP1 binding sites. Furthermore, the Gal4 fusion proteins also activated transcription from a pUC18 vector fragment containing several putative AP1 binding sites. Finally, our results indicate that Gal4 activator proteins binding to Gal4 binding sites and to DNA bound AP1 factors can co-operatively activate transcription.
Asunto(s)
ADN/metabolismo , Proteínas Fúngicas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas de Saccharomyces cerevisiae , Factor de Transcripción AP-1/metabolismo , Dedos de Zinc , Secuencia de Bases , ADN de Hongos/metabolismo , Proteínas de Unión al ADN , Proteínas Fúngicas/química , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
Adenovirus E1A encodes two major proteins of 289 and 243 amino acids (289R and 243R), which both have transcription regulatory properties. E1A-289R is a transactivator whereas E1A-243R primarily functions as a repressor of transcription. Here we show that E1A repression is not restricted to RNA polymerase II genes but also includes the adenovirus virus-associated (VA) RNA genes. These genes are transcribed by RNA polymerase III and have previously been suggested to be the target of an E1A-289R-mediated transactivation. Surprisingly, we found that during transient transfection both E1A proteins repressed VA RNA transcription. E1A repression of VA RNA transcription required both conserved regions 1 and 2 and therefore differed from the E1A-mediated inhibition of simian virus 40 enhancer activity which primarily required conserved region 1. The repression was counteracted by the E1B-19K protein, which also, in the absence of E1A, enhanced the accumulation of VA RNA. Importantly, we show that efficient VA RNA transcription requires expression of both E1A and the E1B-19K protein during virus infection.
Asunto(s)
Proteínas E1A de Adenovirus/farmacología , Regulación Viral de la Expresión Génica , ARN Polimerasa III/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Animales , Chlorocebus aethiops , Análisis Mutacional de ADN , Células HeLa , Humanos , ARN Polimerasa II/antagonistas & inhibidores , ARN Mensajero/genética , ARN Viral/genética , Proteínas Represoras/genética , Relación Estructura-Actividad , Activación TranscripcionalRESUMEN
Varying capacities for autonomous replication have been obtained with bovine papillomavirus type 1 (BPV-1)-based expression vectors in mouse C127 cells. Both integration of the vector DNA into the genome of the host cell and replication as monomeric extrachromosomal elements have been observed. In this study, we have examined what features of BPV-1 vectors influence their replication potential. Transfection of the entire BPV-1 genome into C127 cells resulted in the replication of extrachromosomal monomeric BPV-1 elements. The same result was obtained when a plasmid sequence was inserted into the BPV-1 DNA. However, introduction of foreign, transcriptionally active units resulted in chromosomal integration of the expression vectors. This result was obtained with clones isolated by co-transfection followed by neomycin selection, as well as with clones isolated from neoplastic foci. Supertransfection of a BPV-1-based expression vector into cells harbouring unintegrated replicating BPV-1 genomes resulted in integration of the vector DNA, whereas replication of the resident BPV-1 genomes was unaffected. Extrachromosomal replication of such a vector was achieved when the enhancer and promoter region of the foreign gene were deleted.