To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) association with RBP-J is essential for regulation of virus and cell gene transcription and B lymphocyte transformation into infinitely proliferating lymphoblastoid cells (LCLs). To identify EBNA2-regulated cell genes in LCLs, an EBV recombinant that expresses EBNA2 with its C terminus fused in frame to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor (E2HTF) was used to transform primary B lymphocytes to LCLs. In the presence of 4HT, E2HTF expression level and effects on the LMP1 promoter in transfected BJAB lymphoblasts were similar to EBNA2. In 4HT-supplemented medium, E2HTF EBV recombinant infected LCLs were also similar to EBNA2 LCLs in outgrowth but required higher serum and a restricted range of cell concentrations for consistent growth. In medium without 4HT, E2HTF localized to the cytoplasm, c-myc levels substantially decreased within 6 h, cells stopped growing, and levels of other EBNAs and LMP1 remained stable for 24 h. Over this 24-h period, 30 cell RNAs decreased 2-fold, and 51 other RNAs decreased 1.5-fold. These RNAs encode proteins important in cell adhesion or signaling, transcription, RNA processing, cell-cycle regulation, and survival. Real-time RT-PCR confirmed EBNA2-dependent expression of eight RNAs. RBP-J ͞CBF1 ͉ c-mycI n primary human infection, Epstein-Barr virus (EBV) establishes a latency III (LTIII) infection in B lymphocytes, which is characterized by expression of EBV-encoded nuclear antigens (EBNAs), integral membrane proteins (LMP1 and LMP2), small RNAs (EBERs), and Bam A rightward transcripts. LTIII-infected cell lymphoproliferation is limited by T cell immune responses to EBNAs and LMPs (1, 2). EBNA2 and EBNALP are the first EBV proteins expressed in B lymphocyte infection (1). EBNALP and EBNA2 enhance their upstream promoters, Cp or Wp, which results in transcription of not only EBNALP and EBNA2 but also EBNA3A, EBNA3B, EBNA3C, and EBNA1 (for review see ref. 1). EBNA2 and EBNALP also turn on the EBV LMP1, LMP2B, and LMP2A promoters. EBNA2 directly or indirectly up-regulates B lymphocyte CD21, CD23, c-myc, AML2, BATF, IL16, IL18r, c-fgr, cyclin D2, cdk4, TNF-␣, lymphotoxin, HES1, and G-CSF RNAs (1, 3-7).EBNA2 stably associates with the sequence-specific DNA binding protein RBP-J ͞CBF1 (1), which has at least one cognate site within 500 bp upstream of the Cp, LMP1, LMP2A, and CD23 promoters and within the first intron of the CD21 promoter. EBNALP coactivates with EBNA2 by interaction with the EBNA2 acidic transcriptional activation domain, association with HA95 and PKA, and displacement of HP1␣ from PML bodies (1,(8)(9)(10). The EBNA2 acidic transcriptional activation domain recruits basal and activation-related transcription factors, TAF40, TFIIB, TFIIH, p300͞CBP, PCAF histone acetyltransferases, and a p100 transcription coactivator to promoters (1, 11). The EBNA2 RBP-J association and acidic activating domains are essential for B lymphocyte conversion to lymphoblastoid cells (LCLs) (1). Furthermore, EBNA3A polypept...

Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-J/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three-to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-J, did not change EBNA3C association with RBP-J or EBNA or LMP1 expression, decreased EBNA2 association with RBP-J, decreased c-myc expression, and caused G 0 /G 1 growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c-myc-induced apoptosis. In assays of EBNA2-and RBP-J-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-J binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c-myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-J binding domain. The dependence of EBNA3A effects on the core RBP-J interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-J association strongly implicates RBP-J in c-myc promoter regulation.Epstein-Barr virus (EBV) is a gammaherpesvirus that causes lymphoproliferative diseases in immune-compromised people and rarely in otherwise healthy people (43). EBV infection of primary human B lymphocytes results in EBV expression of nuclear proteins EBNA1, -2, -LP, -3A, -3B, and -3C, integral membrane proteins LMP1, -2A, and -2B, small RNAs (EBERs), BamHI A rightward transcripts that may encode three proteins, and perpetual lymphoblastoid cell growth (lymphoblastoid cell lines [LCLs]) (43, 64). Recombinant reverse genetic analyses indicate that EBNA1, -2, -3A, -3C, and -LP and LMP1 are necessary for LCL outgrowth and that most of the rest of the viral genome is not required. EBNA2 (E2) and EBNALP are the first viral gene products expressed in latent infection, and they coactivate transcription from a subset of cell and viral promoters (1, 28). E2 selfassociates, associates with the sequence-specific DNA binding protein RBP-J/CBF-1/CSL, and activates transcription from promoters containing RBP-J (RBP) binding sites (27,29,32,38,39,50,71,77). The E2 acidic activation domain interacts with transcription factors including TFI...