Epstein-Barr Virus EBNA-3C Is Targeted to and Regulates Expression from the Bidirectional LMP-1/2B Promoter

Abstract

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is essential for EBV-mediated immortalization of human B lymphocytes and regulates both the cell cycle and transcription. Transient reporter gene assays have implicated a pivotal role for EBNA-3C in the regulation of transcription of the majority of latency-associated genes expressed during the EBV growth program, including the viral oncoprotein LMP-1. To examine the regulation of latency gene expression by EBNA-3C, we generated an EBV-positive cell line that inducibly expresses EBNA-3C. This cell line allowed us to examine expression from the endogenous latency gene promoters in the context of an actual latent infection and the presence of other EBNA proteins, in particular EBNA-2, which is presumed to coregulate transcription with EBNA-3C. EBNA-3C induced the expression of both LMP-1 and LMP-2B mRNAs from the bidirectional LMP-1/LMP-2B promoter. In contrast, no effect was seen on expression from the common EBNA promoter Cp, which is responsive to EBNA-3C in reporter assays. Activation of LMP expression was not the consequence of increases in EBNA-2, PU.1 or Spi-B transcription factors, all of which are believed to be critical for activation of LMP-1. Chromatin immunoprecipitation assays furthermore indicated that EBNA-3C is present at the bidirectional LMP-1/LMP-2B promoter. These results indicate that EBNA-3C directly activates the expression of LMP-1 and LMP-2B but is unlikely to significantly regulate EBNA expression via Cp under normal growth conditions. Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is one of only six viral proteins known to be essential for EBV to immortalize primary human B lymphocytes (9,22,33,42,52,72). Although the EBNA-3 genes (3A, 3B, and 3C) probably arose through gene duplication, the sequences of the EBNA-3 proteins are highly divergent, and the fact that both EBNA-3A and EBNA-3C (but not EBNA-3B) are essential for EBVmediated immortalization suggests that their functions have also diverged. These proteins do have related functions, however, regulating both cell cycle progression and transcription.

Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

“…Importantly, although previous reports have detected EBNA 3C association with the EBNA 2-and 3C-regulated viral LMP1 promoter (16), our study provides the first demonstration of EBNA 3C association with cellular DNA elements. We mapped EBNA 3C binding sites at the ITGB1 and ITGA4 promoters and confirmed an additional binding site upstream of ITGB1.…”

Two Epstein–Barr virus (EBV) oncoproteins cooperate to repress expression of the proapoptotic tumour-suppressor Bim: clues to the pathogenesis of Burkitt's lymphoma

“…Although it has been reported that EBNA3C might be necessary to activate LMP1 expression efficiently (Rosendorff et al, 2004;Jimenez-Ramirez et al, 2006), no consistent reduction in LMP1 was detected when EBNA3C was absent from these cells (for example, EBNA3C KO (4) in Figure 1c and also 3CKO (2) in BL2 cells, shown in Figure 5b, have as much or more LMP1 than the revertant carrying lines). Furthermore, the expression levels of two anti-apoptotic proteins (Bcl-2 and A20)-that can be upregulated by LMP1 in BL cells (Henderson et al, 1991;Laherty et al, 1992)remained relatively constant irrespective of whether EBNA3A, 3B or 3C were expressed (see, for example, EBNA3C KO (1) in Figure 1d).…”

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

“…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). Furthermore, EBNA3C amino acids 365-545 or 724-826 repress or activate transcription when tethered to DNA by the Gal4 DNA binding domain (27,28).…”