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Journal of Virology, October 2006, p. 10139-10150, Vol. 80, No. 20
0022-538X/06/$08.00+0     doi:10.1128/JVI.00854-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

EBNA-3B- and EBNA-3C-Regulated Cellular Genes in Epstein-Barr Virus-Immortalized Lymphoblastoid Cell Lines

Adrienne Chen, Bo Zhao, Elliott Kieff, Jon C. Aster, and Fred Wang^*

Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received 26 April 2006/ Accepted 26 July 2006

The cellular pathways that Epstein-Barr virus (EBV) manipulates in order to effect its lifelong persistence within hosts and facilitate its transmission between hosts are not well understood. The EBV nuclear antigen 3 (EBNA-3) family of latent infection proteins consists of transcriptional regulators that influence viral and cellular gene expression in EBV-infected cells. To identify EBNA-3B- and EBNA-3C-regulated cellular genes potentially important for virus infection in vivo, we studied a lymphoblastoid cell line (LCL) infected with an unusual EBV mutant, where a genetic manipulation to delete EBNA-3B also resulted in a significant decrease in EBNA-3C expression and slower than normal growth (3B^–/3C^low). Transcriptional profiling was performed on the 3B^–/3C^low LCLs, and comparison of mutant and wild-type LCL profiles resulted in a group of 21 probe sets representing 16 individual genes showing statistically significant differences in expression. Further quantitative reverse transcription-PCR analyses comparing 3B^–/3C^low LCLs to a previously described EBNA-3B mutant (3B^–) where EBNA-3C expression was normal revealed three potential EBNA-3B-repressed genes, three potential EBNA-3C-repressed genes, and two potential EBNA-3C-activated genes. The most highly EBNA-3C-repressed gene was Jagged1, a cell surface ligand and inducer of the Notch receptor signaling pathway that is usurped by EBV genes essential for B-cell immortalization. 3B^–/3C^low LCLs expressed increased levels of Jagged1 protein and were able to more efficiently induce functional Notch signaling, and this signaling was dependent on Notch cleavage by -secretase. However, inhibiting -secretase-mediated Notch cleavage did not rescue 3B^–/3C^low LCL growth, suggesting that EBNA-3C-mediated repression of this signaling pathway did not contribute to LCL growth in tissue culture. Similarly, expression of the chemokine receptor CXCR4 was reproducibly upregulated in EBNA-3B-null LCLs. Since deletion of EBNA-3B has no significant impact on B-cell immortalization in tissue culture, this finding suggested that EBNA-3B-mediated regulation of CXCR4 may be an important viral strategy for alteration of B-cell homing in the infected host. These studies identify two cellular genes that do not contribute to EBV-induced B-cell growth but whose expression levels are strongly EBNA-3 regulated in EBV-infected primary B cells. These EBV-manipulated cellular pathways may be important for virus survival or transmission in humans, and their independence from EBV-induced B-cell growth makes them potential targets for testing in vivo with the rhesus lymphocryptovirus animal model for EBV infection.

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* Corresponding author. Mailing address: Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115. Phone: (617) 525-4258. Fax: (617) 525-4257. E-mail: fwang{at}rics.bwh.harvard.edu.

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Journal of Virology, October 2006, p. 10139-10150, Vol. 80, No. 20
0022-538X/06/$08.00+0     doi:10.1128/JVI.00854-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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