EBNA-3B- and EBNA-3C-regulated cellular genes in Epstein-Barr virus-immortalized lymphoblastoid cell lines.
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 gamma-secretase. However, inhibiting gamma-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.
Chen
Adrienne
A
Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115, USA.
Zhao
Bo
B
Kieff
Elliott
E
Aster
Jon C
JC
Wang
Fred
F
D000956:Antigens, Viral / Q000235:genetics / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000502:physiology*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D000072100:Jagged-1 Protein; D008214:Lymphocytes / Q000378:metabolism* / Q000821:virology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000032:analysis / Q000235:genetics; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000072099:Serrate-Jagged Proteins
D000956:Antigens, Viral; D002135:Calcium-Binding Proteins; C422762:EBNA-3B antigen; C498774:EBNA-3C, epstein-barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D019718:Receptors, CXCR4; D000072099:Serrate-Jagged Proteins; D012313:RNA