Identification and characterization of CCAAT enhancer-binding protein (C/EBP) as a transcriptional activator for Epstein-Barr virus oncogene latent membrane protein 1.
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.
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681; Department of Oncology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603.
Chieko
C
Noda
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681.
Takayuki
T
Murata
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681.
Teru
T
Kanda
Research Center for Infection-associated Cancer, Institute for Genetic Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo 060-0815, Japan.
Hironori
H
Yoshiyama
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681.
Atsuko
A
Sugimoto
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681.
Daisuke
D
Kawashima
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681.
Shinichi
S
Saito
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681.
Hiroki
H
Isomura
Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681; Department of Oncology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603. Electronic address: ttsurumi@aichi-cc.jp.
Tatsuya
T
Tsurumi
D020816:Amino Acid Motifs; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D005786:Gene Expression Regulation; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D009857:Oncogenes; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014763:Viral Matrix Proteins / Q000235:genetics*
D022762:CCAAT-Enhancer-Binding Proteins; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D012333:RNA, Messenger; D014763:Viral Matrix Proteins; D049452:Green Fluorescent Proteins