RESUMO
The hepatitis B virus (HBV) X gene product trans-activates viral and cellular genes. The X protein (pX) does not bind independently to nucleic acids. The data presented here demonstrate that pX entered into a protein-protein complex with the cellular transcriptional factors CREB and ATF-2 and altered their DNA binding specificities. Although CREB and ATF-2 alone did not bind to the HBV enhancer element, a pX-CREB or pX-ATF-2 complex did bind to the HBV enhancer. Thus, the ability of pX to interact with cellular factors broadened the DNA binding specificity of these regulatory proteins and provides a mechanism for pX to participate in transcriptional regulation. This strategy of altered binding specificity may modify the repertoire of genes that can be regulated by transcriptional factors during viral infection.
Assuntos
Proteínas de Ligação a DNA/farmacologia , DNA/metabolismo , Transativadores/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Sinergismo Farmacológico , Eletroforese em Gel de Poliacrilamida , Regulação Viral da Expressão Gênica , Genes Virais , Humanos , Ligação Proteica , Fatores de Transcrição/farmacologia , Ativação Transcricional , Proteínas Virais Reguladoras e AcessóriasRESUMO
Cytoplasmic RNA was isolated from varicella-zoster virus (VZV)-infected cells. By oligo(dT)-cellulose chromatography, the RNA was separated into polyadenylated, poly (A)+, and nonpolyadenylated, poly (A)-, fractions. RNA blot hybridization was employed to detect and map VZV transcripts. As VZV infection cannot be coordinated, cytoplasmic RNA was isolated from VZV-infected cells when the cells showed extensive cytopathology. Therefore, while the VZV transcripts represented heterogeneous temporal classes, it may be assumed that late VZV RNA predominated. At least 41, and as many as 67 (depending on DNA probe overlap), VZV polyadenylated transcripts have been identified. Preliminary evidence for the presence of two VZV-specific nonpolyadenylated, cytoplasmic transcripts was observed.
Assuntos
Herpesvirus Humano 3/genética , RNA Mensageiro/genética , RNA Viral/genética , Mapeamento Cromossômico , DNA Viral/genética , Genes Virais , Poli A/genética , RNA/genética , RNA Neoplásico/genética , Transcrição GênicaRESUMO
Several viral transcriptional activators have been shown to interact with the basal transcription factor TATA-binding protein (TBP). These associations have been implicated in facilitating the assembly of the transcriptional preinitiation complex. We report here that the hepatitis B virus protein X (pX) specifically binds to TBP in vitro. While truncations of the highly conserved carboxyl terminus of TBP abolished this binding, amino-terminal deletions had no effect. Deletion analysis suggests that a domain consisting of 71 aa in the highly conserved carboxyl-terminal region of TBP is necessary for its interaction with pX. The minimal region in pX sufficient for its interaction with TBP includes aa 110-143. Furthermore, TBP from phylogenetically distinct species including Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, and Solanum tuberosum (potato) bound to pX. The pX-TBP interaction was inhibited in the presence of nonhydrolyzable analogs of ATP, suggesting a requirement for ATP. These results provide an explanation for the promiscuous behavior of pX in the transactivation of a large repertoire of cellular promoters. This study further implicates a fundamental role for pX in modulating transcriptional regulatory pathways by interacting with the basal transcription factor TBP.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Antígenos da Hepatite B/metabolismo , TATA Box , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Reagentes de Ligações Cruzadas , Dados de Sequência Molecular , Testes de Precipitina , Proteína de Ligação a TATA-Box , Proteínas Virais Reguladoras e AcessóriasRESUMO
Lack of an in vitro culture system for human hepatitis B virus has hampered the ability to address fundamental questions regarding the viral life cycle and the effect of viral gene products during productive infection. To study the activity of HBV X protein (HBx) in the context of a viral infectious cycle, we provided HBx in trans during adenovirus infection of liver-derived cells. In hepatoma cells infected with adenovirus mutants deficient in expression of various E1A products, HBx was able to partially substitute for the transcriptional activation function of E1A. HBx also activated adenovirus replication, but to a lesser extent than the activation of transcription. Adenovirus genes transcribed by either RNA polymerase II or RNA polymerase III were activated by HBx during infection. These results suggest that HBx and E1A activate transcription by a similar mechanism and that this viral infection system will be useful for characterization of the functional activities of HBx.
Assuntos
Proteínas E1 de Adenovirus/fisiologia , Vírus da Hepatite B/fisiologia , Transativadores/fisiologia , Ativação Transcricional , Proteínas E1 de Adenovirus/genética , Replicação do DNA/genética , DNA Viral/genética , Deleção de Genes , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Humanos , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Transativadores/genética , Células Tumorais Cultivadas , Proteínas Virais Reguladoras e AcessóriasRESUMO
The liver-specific enhancer I of the human hepatitis B virus contains several regions of DNA-protein interaction. Located within this element are also the domains of a promoter controlling the synthesis of the X open reading frame. Functional domains of the enhancer I and the X gene promoter were identified using DNase I protection analysis, deletion mutagenesis, and cell transfections. A unique liver-specific interaction was identified within this element whose binding site includes a direct sequence repeat, 5'-AGTAAACAGTA-3'. The factor(s) binding to this sequence motif was purified by oligonucleotide-affinity chromatography. Binding of this factor appears to play a key role in determining the overall enhancer function. Additionally, the interaction of several purified factors is presented. Cotransfection of liver cells with expression vectors encoding transcriptional factors resulted in trans-activation of the promoter/enhancer function. Based on the results of genetic analysis a model outlining the functional domains of the enhancer/promoter region is presented.
Assuntos
Elementos Facilitadores Genéticos , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Proteínas Estimuladoras de Ligação a CCAAT , Análise Mutacional de DNA , DNA Viral/genética , Proteínas de Ligação a DNA/fisiologia , Genes Virais , Humanos , Técnicas In Vitro , Fígado/fisiologia , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/fisiologia , Transcrição Gênica , Proteínas Estruturais Virais/genéticaRESUMO
The hepatitis B virus X gene product trans activates transcription from a variety of viral and cellular regulatory elements. We expressed the complete, nonfused X protein in Escherichia coli and showed it to be active in trans activating a human immunodeficiency virus long terminal repeat-linked chloramphenicol acetyltransferase reporter gene.