RESUMO
Both interleukin-4 (IL-4) and interleukin-13 (IL-13) induce the transcription factor NF-IL4 (nuclear factor IL-4) which preexists in an inactive form and binds to an IL-4 responsive element (IL-4RE) in the promoter regions of IL-4/IL-13-dependent genes. UV-crosslinking and SDS gel electrophoresis indicate that NF-IL4 consists of at least two DNA-binding components of 50 kDa and 100-130 kDa. The IL-4 responsive element is also recognized by an interferon-gamma (IFN-gamma)-induced DNA binding protein for which a Mr of 50 kDa has been determined. A common DNA binding motif for different transcription factors might provide the basis for the frequently observed functional antagonism between IL-4/IL-13 and IFN-gamma. The activation of transcription factors by IL-4/IL-13 and IFN-gamma could be blocked by inhibitors of tyrosine kinases and ser/thr phosphatases but not by a PKC inhibitor, suggesting related signal transduction pathways for these cytokines.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-13/farmacologia , Interleucina-4/farmacologia , Regiões Promotoras Genéticas , Fatores de Transcrição/biossíntese , Transcrição Gênica/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/metabolismo , Transfecção , Raios UltravioletaRESUMO
The effects of interleukin-13 (IL-13) and interleukin-4 (IL-4) on cellular functions were shown to be quite similar. We provide evidence that in monocytes as well as in T lymphocytes both IL-4 and IL-13 activate the same recently identified transcription factor NF-IL4 which binds to the specific responsive element IL-4RE. In addition, we show that a nuclear factor activated by interferon-gamma also interacts with the IL-4RE. It differs from NF-IL4 in the electrophoretic mobility of the complex with DNA, in its DNA-binding specificity and in the proteins interacting with the DNA sequence. Sensitivity against various enzyme inhibitors suggests that components of the signal transduction pathway are shared by all three cytokines.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon gama/farmacologia , Interleucina-4/farmacologia , Interleucinas/farmacologia , Monócitos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Células CHO , Núcleo Celular/metabolismo , Células Cultivadas , Cricetinae , Humanos , Interleucina-13 , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Oligodesoxirribonucleotídeos , Regiões Promotoras Genéticas , Receptores de IgE/genética , Receptores de IgG/genética , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial cells.
Assuntos
Linfócitos B/microbiologia , Linfoma de Burkitt/microbiologia , Regulação Viral da Expressão Gênica , Genes Virais/genética , Herpesvirus Humano 4/genética , Proteínas Imediatamente Precoces/genética , Linhagem Celular , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Proteínas Recombinantes/biossíntese , Vaccinia virus/genéticaRESUMO
Lytic transition of Epstein-Barr virus (EBV) is initiated by distinct immediate early regulators of the viral cycle, in synchronization to temporary, permissive conditions during host cell differentiation. We developed eukaryotic vectors suitable to imitate the processes involved in lytic transition in cell culture systems. Two stable B cell lines were established: R59Z activator cells were used to induce lytic EBV expression in a constitutive manner by the production of the BZLF 1 trans-activator (Zta). R7-57 reporter cells, on the other hand, signaled induced activity of the lytic origin of EBV replication (ori Lyt). Different modes, like chemical induction, lytic superinfection with EBV and single gene trans-activation converted the recombinant ori Lyt element in R7-57 reporter cells. BZLF 1, transiently expressed in R7-57 reporter cells, was the only EBV trans-activator found, sufficient in inducing the viral lytic cycle. Basing on these experiments, trans-cellular activation of EBV was tested by cocultivation of BZLF 1-expressing R59Z activator cells with the R7-57 reporter line. No lytic effect on the reporter cells could be measured, neither by cocultivation of activator cells nor by coincubation of BZLF 1-containing cell lysates. Latency breaking activity, however, was transferred from activator to reporter cells when active, exogenous virus was added. The cell system described in these experiments provides a tool for the detection of EBV reactivation and demonstrates the potential of the lytic regulatory gene BZLF 1.
Assuntos
Linfócitos B , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 4/crescimento & desenvolvimento , Transativadores/genética , Proteínas Virais/genética , Animais , Callithrix , Linhagem Celular , Herpesvirus Humano 4/genética , Humanos , Recombinação Genética , Transfecção , Células Tumorais Cultivadas , Ativação ViralRESUMO
We have analyzed the activity and regulated expression of a new Epstein-Barr virus (EBV) trans-activator (I'ta) encoded by left reading frame 4 (BI'LF4) of the BamHI I'fragment. The gene was detected in all genomes of established EBV strains and individual isolates, with the exception of B95-8, where the type-specific deletion of this open reading frame is tolerated in vitro. Specific trans-activation of two EBV promoters (early MS and I'ta promoter) could be shown in cotransfection assays. The I'ta product affected autoactivation but had no influence on heterologous target promoters. The I'ta promoter segment was shown to be costimulated in the process of host cell differentiation in the absence of other EBV gene products. Expression of the reading frame in bacteria identified a 48-kDa protein as a stable gene product. I'ta-specific antibodies were detected in sera from EBV-positive persons (nasopharyngeal carcinoma). When expressed with suitable eucaryotic vectors, a nuclear protein could be immunostained in transfected cells. Our experiments suggest a cell type-specific requirement for I'ta in the lytic cycle of EBV at a determined differentiation stage of the host cell.
Assuntos
Diferenciação Celular , Genes Virais , Herpesvirus Humano 4/genética , Transativadores/genética , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cloranfenicol O-Acetiltransferase/genética , Clonagem Molecular , Imunofluorescência , Regulação Viral da Expressão Gênica , Células HeLa/citologia , Antígenos de Superfície da Hepatite B/genética , Humanos , Camundongos , Fases de Leitura , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , TeratomaRESUMO
A method is described for the identification of type A and type B isolates of Epstein-Barr virus (EBV) by means of the polymerase chain reaction. The use of three pairs of primers specific for genomic sequences coding for the two forms of EBV nuclear antigen (EBNA), 2A and 2B, and for a DNA sequence from the BamZ/BamR region allows the reliable and rapid detection of type A and B viruses in as little as 1000 EBV positive cells.
Assuntos
DNA Viral/química , Herpesvirus Humano 4/genética , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase , Antígenos Nucleares , Antígenos Virais/genética , Sequência de Bases , Linhagem Celular , Genes Virais , Herpesvirus Humano 4/imunologia , Humanos , Dados de Sequência MolecularRESUMO
A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.
