The Epstein-Barr Virus Immortalizing Protein EBNA-2 is Targeted to DNA by a Cellular Enhancer-Binding Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-10, Vol.90 (20), p.9237-9241
• Main Authors: Ling, Paul D., Rawlins, Dan R., Hayward, S. Diane
• Format: Article
• Language: English
• Subjects: Antigens, Viral - metabolism; B lymphocytes; Base Sequence; Binding Sites; Biochemistry; Biological and medical sciences; Cell lines; Cell Nucleus - metabolism; Deoxyribonucleic acid; Deoxyribonucleoproteins - chemistry; DNA; DNA - metabolism; DNA-Binding Proteins - metabolism; Enhancer Elements, Genetic; Epstein Barr virus infections; Epstein-Barr virus; Epstein-Barr Virus Nuclear Antigens; Fundamental and applied biological sciences. Psychology; Genetic mutation; Herpesvirus 4, Human - genetics; Human herpesvirus 4; In Vitro Techniques; Microbiology; Molecular Sequence Data; Nuclear Proteins - metabolism; Oligonucleotide probes; Oligonucleotides; Promoter Regions, Genetic; Proteins; Replicative cycle, interference, host-virus relations, pathogenicity, miscellaneous strains; Sequence Alignment; Sequence Homology, Nucleic Acid; Transactivation; Virology; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.20.9237

The Epstein-Barr virus nuclear antigen EBNA-2 is essential for Epstein-Barr virus-induced immortalization of B cells. EBNA-2 is a transcriptional activator capable of modifying the expression of specific viral and cellular genes. However, the mechanism of EBNA-2 transactivation has been an enigma. We used a fractionated extract of CA46 lymphoblastoid cells and bacterially expressed EBNA-2 polypeptides to demonstrate that EBNA-2 is targeted to the Epstein-Barr virus latency C promoter (Cp) through interaction with a cellular DNA binding protein designated Cp binding factor 1 (CBF1). A glutathione S-transferase-EBNA-2 fusion protein containing aa 252-425 of EBNA-2 interacted with CBF1 to yield a slowly migrating complex in an electrophoretic mobility shift assay. Mutation of EBNA-2 aa 323 and 324, which lie within a highly conserved amino acid motif, abolished the interaction with CBF1. This same mutation also abolished the ability of EBNA-2 to activate the Cp in a cotransfection assay. The binding site for CBF1 was localized to residues -359 to -388 of the Cp by using an electrophoretic mobility shift assay and DNase I footprinting. Introduction of multiple copies of the CBF1 binding site upstream of a minimal heterologous promoter conferred EBNA-2 responsiveness on that promoter. Mutation of a core sequence CNGTGGGAA abolished CBF1 binding, and the mutated sequence was unable to mediate EBNA-2 transactivation. The CBF1 core sequence also occurs in other EBNA-2-responsive promoters suggesting that CBF1 may mediate EBNA-2 transactivation of both cellular and viral targets.