EBNA-2 upregulation of Epstein-Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1.
The EBNA-2 protein is essential for the establishment of a latent Epstein-Barr virus (EBV) infection and for B-cell immortalization. EBNA-2 functions as a transcriptional activator that modulates viral latency gene expression as well as the expression of cellular genes, including CD23. We recently demonstrated that EBNA-2 transactivation of the EBV latency C promoter (Cp) is dependent on an interaction with a cellular DNA-binding protein, CBF1, for promoter targeting. To determine whether targeting via CBF1 is a common mechanism for EBNA-2-mediated transactivation, we have examined the requirements for activation of the cellular CD23 promoter. Binding of CBF1 to a 192-bp mapped EBNA-2-responsive region located at position -85 bp to -277 bp upstream of the CD23 promoter was detected in electrophoretic mobility shift assays. The identity of the bound protein as CBF1 was established by showing that the bound complex was competed for by the CBF1 binding site from the EBV Cp, that the bound protein could be supershifted with a bacterially expressed fusion protein' containing amino acids 252 to 425 of EBNA-2 but was unable to interact with a non-CBF1-binding EBNA-2 mutant (WW323SR), and that in UV cross-linking experiments, the Cp CBF1 binding site and the CD23 probe bound proteins of the same size. The requirement for interaction with CBF1 was demonstrated in a transient cotransfection assay in which the multimerized 192-bp CD23 response region was transactivated by wild-type EBNA-2 but not by the WW323SR mutant. Reporter constructions carrying multimerized copies of the 192-bp CD23 response region or multimers of the CBF1 binding site from the CD23 promoter were significantly less responsive to EBNA-2 transactivation than equivalent constructions carrying a multimerized region from the Cp or multimers of the CBF1 binding site from the Cp. Direct binding and competition assays using 30-mer oligonucleotide probes representing the individual CBF1 binding sites indicated that CBF1 bound less efficiently to the CD23 promoter and the EBV LMP-1 promoter sites than to the Cp site. To investigate the basis for this difference, we synthesized a series of oligonucleotides carrying mutations across the CBF1 binding site and used these as competitors in electrophoretic mobility shift assays. The competition experiments indicated that a central core sequence, GTGGGAA, common to all known EBNA-2-responsive elements, is crucial for CBF1 binding. Flanking sequences on either side of this core influence the affinity for CBF1.(ABSTRACT TRUNCATED AT 400 WORDS)
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Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Hsieh
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JJ
Ruf
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Rawlins
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Hayward
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D000956:Antigens, Viral / Q000502:physiology*; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D002467:Cell Nucleus / Q000737:chemistry; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000502:physiology*; D019309:Epstein-Barr Virus Nuclear Antigens; D005656:Fungal Proteins / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D017455:Receptors, IgE / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D015533:Transcriptional Activation; D014763:Viral Matrix Proteins / Q000235:genetics*; D017735:Virus Latency
D000956:Antigens, Viral; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C064042:CBF1 protein, S cerevisiae; D017931:DNA Primers; D004268:DNA-Binding Proteins; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D005656:Fungal Proteins; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D017455:Receptors, IgE; D029701:Saccharomyces cerevisiae Proteins; D014763:Viral Matrix Proteins