RNAs induced by Epstein–Barr virus nuclear antigen 2 in lymphoblastoid cell lines

Abstract

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 antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16 INK4A and p14 ARF expression

“…Certainly, constitutive EBNA2-mediated c-myc up-regulation has a central role in EBV-mediated LCL growth (49,62) and almost certainly underlies p16 INK4A and p14 ARF induction in EBV-infected B cells in the absence of EBNA3C or EBNA3A. Forced MYC expression induces p16 INK4A and p14 ARF in normal human diploid fibroblasts and megakaryocytes (42,63).…”

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

“…2). (ii) Several potentially important genes for LCL cell growth or survival changed with FDR < 0.05 significance or with <1.5-fold effect. KLHL6 HSPA1A ENO2 CST3 PON2 ALDOC RAB13 IFI27 CYP1B1 SLC2A3 P4HA2 SERPINA1 SOX9 LMNB1 TBC1D4 MAL DUSP2 MNDA CRIP1 HMGCS1 ARHGAP6 XCL1 LTA CCL22 CD24 CXCL12 ASCL1 LGALS8 CXCR4 FRMD4B TUBB2B S100A6 OAS3 C7orf68 TMEM45A EVC STAG3 NOD2 BNIP3L TMEM120A IL17RB TMEM107 AICDA C5orf32 CNN3 PFKFB4 TOX2 GPR133 LOC285628 MAFF GIMAP1 RAP1A FAM120A SCARB2 RRM2 TRIB1 ARHGAP25 AMMECR1 HIST1H4C EIF4G1 ATRX HIST1H1C LYN AKAP9 LHX2 BAT2D1 MTUS1 PSME4 QKI IGJ ZNF3 ANKMY2 IQGAP1 SFRS2IP ANKRD36 HIST1H2AC TPR GIMAP5 ANKRD12 BZW2 ARL5A ZNF281 CHST12 CCDC88A KLF13 CLEC4A YTHDF2 THRAP3 FAM46C AFF3 GIMAP7 BTNL9 DST CHD9 SGOL2 RIF1 KBTBD8 NIPBL Although EBNA2 has a much larger initial effect on MYC upregulation in LCLs, EBNA3C 1.3-fold up-regulation could be important at a specific point in cell cycle or under specific growth conditions, as has been described for the EBNA3C effect on EBV LMP1 expression in Latency III infected Raji BL cells (9,40,41). Also, TCL1A was EBNA3C up-regulated 1.7-fold, with FDR < 0.05.…”

“…Because EBNA2 and EBNA3A regulation of transcription through RBPJ is essential for LCL growth, we investigated the overlap of EBNA3C and EBNA2 effects on transcript abundance in LCLs (41,51) and in BL cells (52). Notably, all significant EBNA2 effects on LCL RNAs are up-regulatory (41,51). Of the 461 EBNA3C-regulated genes common to HU133A and Agilent arrays used in EBNA2 regulated LCL and BL RNA analyses, 55 RNAs were effected by EBNA3C and EBNA2, P value of <1.7 × 10 −14 ( Fig.…”

Epstein–Barr virus super-enhancer eRNAs are essential for MYC oncogene expression and lymphoblast proliferation

“…Because EBNA2 ChIP-seq peaks mostly overlapped with MYC ESE eRNAs, we tested whether EBNA2 inactivation reduces MYC ESE eRNA levels. We used conditional EBNA2 LCLs wherein EBNA2 is fused to the hormone binding domain of a modified estrogen receptor hormone binding domain that is only responsive to 4-hydroxytamoxifen (4HT) (34). In the presence of 4HT, the LCLs grows normally, whereas in the absence of 4HT, EBNA2 is translocated to the cytoplasm, where it is degraded, causing the LCLs to stop growing.…”