Epstein-barr virus nuclear antigen 3C activates the latent membrane protein 1 promoter in the presence of Epstein-Barr virus nuclear antigen 2 through sequences encompassing an spi-1/Spi-B binding site.
The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) protein is a transcriptional regulator of viral and cellular genes that is essential for EBV-mediated immortalization of B lymphocytes in vitro. EBNA-3C can inhibit transcription through an association with the cellular DNA-binding protein Jkappa, a function shared by EBNA-3A and EBNA-3B. Here, we report a mechanism by which EBNA-3C can activate transcription from the EBV latent membrane protein 1 (LMP-1) promoter in conjunction with EBNA-2. Jkappa DNA-binding sites were not required for this activation, and a mutant EBNA-3C protein unable to bind Jkappa activated transcription as efficiently as wild-type EBNA-3C, indicating that EBNA-3C can regulate transcription through a mechanism that is independent of Jkappa. Furthermore, activation of the LMP-1 promoter is a unique function of EBNA-3C, not shared by EBNA-3A and EBNA-3B. The DNA element through which EBNA-3C activates the LMP-1 promoter includes a Spi-1/Spi-B binding site, previously characterized as an important EBNA-2 response element. Although this element has considerable homology to mouse immunoglobulin light chain promoter sequences to which the mouse homologue of Spi-1 binds with its dimerization partner IRF4, we demonstrate that the IRF4-like binding sites in the LMP-1 promoter do not play a role in EBNA-3C-mediated activation. Both EBNA-2 and EBNA-3C were required for transcription mediated through a 41-bp region of the LMP-1 promoter encompassing the Spi binding site. However, EBNA-3C had no effect on transcription mediated in conjunction with the EBNA-2 activation domain fused to the GAL4 DNA-binding domain, suggesting that it does not function as an adapter between EBNA-2 and the cellular transcriptional machinery. Like EBNA-2, EBNA-3C bound directly to both Spi-1 and Spi-B in vitro. This interaction was mediated by a region of EBNA-3C encompassing a likely basic leucine zipper (bZIP) domain and the ets domain of Spi-1 or Spi-B, reminiscent of interactions between bZIP and ets domains of other transcription factors that result in their targeting to DNA. There are many examples of regulation of the hematopoietic-specific Spi transcription factors through protein-protein interactions, and a similar regulation by EBNA-3C, in conjunction with EBNA-2, is likely to be an important and unique contribution of EBNA-3C to EBV-mediated immortalization.
Zhao
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B
Program in Viral Oncogenesis and Tumor Immunology, Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
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D001665:Binding Sites; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020218:Response Elements; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000235:genetics*; D000099232:Proto-Oncogene Protein Spi-1
D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014763:Viral Matrix Proteins; D000099232:Proto-Oncogene Protein Spi-1; C037662:EBV-associated membrane antigen, Epstein-Barr virus; C076877:SPIB protein, human