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

Abstract

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.

An Update on Epstein-Barr Virus and Nasopharyngeal Carcinogenesis

“…EBNA2, EBNA3C and LMP1 are key in the transformation of EBV-infected cells [3,4]. LMP1 is the main transforming protein of EBV and functions as a classic oncogene in fibroblast transformation assay [5].…”

Epstein–Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines

“…EBNA3CHT LCLs were cultured and transfected with Ori-P based EBNA3C expression vectors (14,16). Migration assays were performed as described (18).…”

“…Also, activated Notch expression can partially substitute for EBNA2 in maintaining LCL growth (10). Surprisingly, EBNA3A and EBNA3C are each uniquely important for initial and continued LCL growth, whereas EBNA3B is not required (11)(12)(13)(14)(15)(16), although EBNA3B also alters cell gene transcription (17,18). EBNA3C uniquely up-regulates LMP1 (19)(20)(21)(22)(23)(24), CD21(CR2), TCL1A, and ITGA4 expression and represses JAG1, NCALD, p16, BIM, and FLNA expression (14-16, 18, 19, 25, 26).…”

Epstein-Barr virus–associated lymphoproliferative disorders

“…Three distinct latency programs that are equivalent to the differentiation state of B cells have been postulated after analysis of EBV-infected B cells in cell lines and seropositive individuals [3,10]. The presence of different EBV latency programs is thought to have evolved in order for EBV-infected cells to survive in the face of an adaptive immune response and surveillance [10,11].…”