Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1.
Expression of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) oncogene is regulated by the EBV nuclear protein 2 (EBNA-2) transactivator. EBNA-2 is known to interact with the cellular DNA-binding protein J kappa and is recruited to promoters containing the GTGGGAA J kappa recognition sequence. The minimal EBNA-2-responsive LMP-1 promoter includes one J kappa-binding site, and we now show that mutation of that site, such that J kappa cannot bind, reduces EBNA-2 responsiveness by 60%. To identify other factors which interact with the LMP-1 EBNA-2 response element (E2RE), a -236/-145 minimal E2RE was used as a probe in an electrophoretic mobility shift assay. The previously characterized factors J kappa, PU.1, and AML1 bind to the LMP-1 E2RE, along with six other unidentified factors (LBF2 to LBF7). Binding sites were mapped for each factor. LBF4 is B- and T-cell specific and recognizes the PU.1 GGAA core sequence as shown by methylation interference. LBF4 has a molecular mass of 105 kDa and is probably unrelated to PU.1. LBF2 was found only in epithelial cell lines, whereas LBF3, LBF5, LBF6, and LBF7 were not cell type specific. Mutations of the AML1- or LBF4-binding sites had no effect on EBNA-2 transactivation, whereas mutation of the PU.1-binding site completely eliminated EBNA-2 responses. A gst-EBNA-2 fusion protein specifically depleted PU.1 from nuclear extracts and bound in vitro translated PU.1, providing biochemical evidence for a direct EBNA-2-PU.1 interaction. Thus, EBNA-2 transactivation of the LMP-1 promoter is dependent on interaction with at least two distinct sequence-specific DNA-binding proteins, J kappa and PU.1. LBF3, LBF5, LBF6, or LBF7 may also be involved, since their binding sites also contribute to EBNA-2 responsiveness.
Johannsen
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E
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts.
Koh
E
E
Mosialos
G
G
Tong
X
X
Kieff
E
E
Grossman
S R
SR
D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D050676:Core Binding Factor Alpha 2 Subunit; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D019309:Epstein-Barr Virus Nuclear Antigens; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D008214:Lymphocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic; D015534:Trans-Activators; D014157:Transcription Factors; D015533:Transcriptional Activation; D014763:Viral Matrix Proteins / Q000235:genetics*
D000956:Antigens, Viral; D050676:Core Binding Factor Alpha 2 Subunit; D017931:DNA Primers; D004268:DNA-Binding Proteins; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C497570:RBPJ protein, human; C493728:RUNX1 protein, human; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D015534:Trans-Activators; D014157:Transcription Factors; D014763:Viral Matrix Proteins; C062022:v-Spi-1 protein, Friend spleen focus-forming virus; D005982:Glutathione Transferase