Abstract
Epstein-Barr virus LMP1, a major oncoprotein expressed in latent infection, is critical for primary B cell transformation, functioning as a TNFR family member by aggregation in the plasma membrane resulting in constitutive activation of cellular signals, such as NF-κB, MAPK, JAK/STAT, and AKT. Although transcription of LMP1 in latent type III cells is generally under the control of the viral coactivator EBNA2, little is known about EBNA2-independent LMP1 expression in type II latency. We thus screened a cDNA library for factors that can activate the LMP1 promoter in an EBNA2-independent manner, using a reporter assay system. So far, we have screened >20,000 clones, and here identified C/EBPε as a new transcriptional activator. Exogenous expression of C/EBPα, -β, or -ε efficiently augmented LMP1 mRNA and protein levels in EBV-positive cell lines, whereas other members of the C/EBP family exhibited modest or little activity. It has been demonstrated that LMP1 gene transcription depends on two promoter regions: proximal (ED-L1) and distal (TR-L1). Interestingly, although we first used the proximal promoter for screening, we found that C/EBP increased transcription from both promoters in latent EBV-positive cells. Mutagenesis in reporter assays and EMSA identified only one functional C/EBP binding site, through which activation of both proximal and distal promoters is mediated. Introduction of point mutations into the identified C/EBP site in EBV-BAC caused reduced LMP1 transcription from both LMP1 promoters in epithelial cells. In conclusion, C/EBP is a newly identified transcriptional activator of the LMP1 gene, independent of the EBNA2 coactivator.
Highlights
Expression mechanism of Epstein-Barr virus (EBV) oncogene latent infection integral membrane protein 1 (LMP1) is not fully understood
We found that Ets domain family transcription factors were frequently isolated: two clones of the hits encode Friend leukemia virus integration 1 (FLI1), and four clones encode PU.1, known as spleen forming virus proviral integration 1 (SPI1)
The CCAAT enhancer-binding protein (C/EBP)⑀ protein was identified by our screening to increase the proximal LMP1 (ED-L1) promoter activity
Summary
Expression mechanism of EBV oncogene LMP1 is not fully understood. Results: C/EBP was newly isolated to enhance the LMP1 promoter in our transient assay system. We screened a cDNA library for factors that can activate the LMP1 promoter in an EBNA2-independent manner, using a reporter assay system. LMP1 is expressed in latent type II EBV infection in Hodgkin disease B lymphocytes and NPC epithelial cells Because it functions as a constitutive TNFR family member by aggregation in the plasma membrane, resulting in constitutive activation of cellular signaling, through NF-B, MAPK, JAK/STAT, and AKT [1,2,3,4], LMP1 is assumed to be the most major oncogene encoded by EBV. We newly cloned the CCAAT enhancer-binding protein (C/EBP) family transcription factor that augments both proximal and distal promoter activation of LMP1 by binding to a motif in the proximal promoter. We constructed a mutant EBV with a point mutation in the C/EBP binding site, and confirmed the importance of binding for LMP1 expression in latent cells
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