Induction of Epstein-Barr Virus Oncoprotein LMP1 by Transcription Factors AP-2 and Early B Cell Factor

Abstract

Latent membrane protein 1 (LMP1) is a major oncogene essential for primary B cell transformation by Epstein-Barr virus (EBV).Previous studies suggested that some transcription factors, such as PU.1, RBP-J, NF-B, and STAT, are involved in this expression, but the underlying mechanism is unclear. Here, we identified binding sites for PAX5, AP-2, and EBF in the proximal LMP1 promoter (ED-L1p). We first confirmed the significance of PU.1 and POU domain transcription factor binding for activation of the promoter in latency III. We then focused on the transcription factors AP-2 and early B cell factor (EBF). Interestingly, among the three AP-2-binding sites in the LMP1 promoter, two motifs were also bound by EBF. Overexpression, knockdown, and mutagenesis in the context of the viral genome indicated that AP-2 plays an important role in LMP1 expression in latency II in epithelial cells. In latency III B cells, on the other hand, the B cell-specific transcription factor EBF binds to the ED-L1p and activates LMP1 transcription from the promoter. IMPORTANCE Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is crucial for B cell transformation and oncogenesis of other EBVrelated malignancies, such as nasopharyngeal carcinoma and T/NK lymphoma.Its expression is largely dependent on the cell type or condition, and some transcription factors have been implicated in its regulation. However, these previous reports evaluated the significance of specific factors mostly by reporter assay. In this study, we prepared point-mutated EBV at the binding sites of such transcription factors and confirmed the importance of AP-2, EBF, PU.1, and POU domain factors. Our results will provide insight into the transcriptional regulation of the major oncogene LMP1. The Epstein-Barr virus (EBV) is a human gammaherpesvirus that mainly infects and establishes latent infection in B lymphocytes, but it can also infect other types of cells, including NK, T, and epithelial cells. EBV infection has been implicated as a causal factor in a variety of malignancies, and the expression pattern of viral latent genes varies depending on the tissue of origin and the state of the tumors. Neoplasms such as Burkitt lymphomas or gastric carcinomas express only EBV-encoded small RNA (EBER) and EBV nuclear antigen 1 (EBNA1) (type I latency), whereas some Hodgkin lymphomas, nasopharyngeal carcinomas (NPC), and NK/T lymphomas express EBER, EBNA1, latent membrane protein 1 (LMP1), and LMP2 genes (type II latency). In addition to the type II genes, EBNA2, EBNA3, and EBNA-LP are also expressed in immunosuppression-related lymphomas or lymphoblastoid cell lines (LCLs; type III latency). LMP1 constitutively activates cellular signaling through NF-B, mitogen-activated protein, JAK/STAT, and AKT and is believed to be a major oncogene encoded by EBV (1-11).Two promoters regulate LMP1 gene transcription, with mechanisms that differ between type II and type III infection. In latency III in B lymphocytes, LMP1 transcription from the proximal ED-L1 promoter is acti...

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells

“…The underlying mechanism that recruits EBNA2 specifically to these sites in B cells is still not understood and hard to study in the constitutive presence of CBF1. Since it was expected and also shown by other labs that CBF1 knock-down is not compatible with long term proliferation of LCLs [16, 17], we used a CBF1 deficient EBV negative B cell line to study whether EBNA2 can activate cellular genes and bind to chromatin in the absence of CBF1. This CBF1 deficient B cell line had been generated by targeted homologous recombination in DG75, a somatic cell line derived from an EBV negative Burkitt's lymphoma [36].…”

“…However, inactivation of the PU.1/SPI1 binding site at the LMP1 promoter in the viral genome did not grossly change the transformation potential of the viral mutants. LMP1 expression and proliferation was diminished but not abolished while inactivation of the EBF1 binding site ablated LMP1 expression [16]. To date, there is no experimental proof indicating that EBNA2 is recruited to chromatin by PU.1/SPI1 [17].…”

“…PU.1/SPI1 DNA binding sites are critical for LMP1 promoter luciferase activation [3, 13–15]. However, its contribution to LMP1 expression in the context of the entire viral genome is surprisingly weak [16]. Most recently it has been shown that EBNA2 enhances the binding of CBF1 and EBF1 to chromatin and EBF1 is critical for expression of the EBNA2 viral target gene LMP1 [16, 17].…”

“…However, its contribution to LMP1 expression in the context of the entire viral genome is surprisingly weak [16]. Most recently it has been shown that EBNA2 enhances the binding of CBF1 and EBF1 to chromatin and EBF1 is critical for expression of the EBNA2 viral target gene LMP1 [16, 17]. By sequential chromatin immunoprecipitation, EBF1 and EBNA2 have been shown to bind to the same chromatin fragment in the same cell [17].…”

DNA Methylation Pattern of Early Genes of Epstein Barr Virus Associated With Gastric Carcinoma in Group of Iraqi Patients

“…The virus has three latency stages: stage one, two and three, by the patterns of the expression of the virus genes. In gastric carcinoma latency stage one or two were responsible of latent infection, which produce several viral oncogenic genes 4 .Viral genes expression modifies relying on the state of the tumors and the tissue of origin 5 . Additionally, the latent gene stage could be inhibiting by methylation.…”