Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors.
BACKGROUND
Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors.


RESULTS
Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies.


CONCLUSIONS
Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.
Owen
Thomas J
TJ
School of Cancer Sciences, University of Birmingham, Birmingham, UK.
O'Neil
John D
JD
Dawson
Christopher W
CW
Hu
Chunfang
C
Chen
Xiaoyi
X
Yao
Yunhong
Y
Wood
Victoria H J
VH
Mitchell
Louise E
LE
White
Robert J
RJ
Young
Lawrence S
LS
Arrand
John R
JR
D051698:Activating Transcription Factor 2 / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D020411:Oligonucleotide Array Sequence Analysis; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012320:RNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019474:Transcription Factors, TFIII / Q000235:genetics / Q000378:metabolism; D000098832:Transcription Factor TFIIIC
D051698:Activating Transcription Factor 2; D019309:Epstein-Barr Virus Nuclear Antigens; C099537:Epstein-Barr virus encoded RNA 1; C106732:Epstein-Barr virus encoded RNA 2; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D012367:RNA, Viral; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D012320:RNA Polymerase III