This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, H.
Right arrow Articles by Hayward, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.
Right arrow Articles by Hayward, S. D.

 Previous Article  |  Next Article 

Journal of Virology, February 2005, p. 1724-1733, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1724-1733.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Regulation of Expression of the Epstein-Barr Virus BamHI-A Rightward Transcripts

Honglin Chen,1 Jian Huang,2,3 Frederick Y. Wu,2 Gangling Liao,2 Lindsey Hutt-Fletcher,4 and S. Diane Hayward2,3*

Department of Microbiology, The University of Hong Kong, Hong Kong,1 Viral Oncology Program, Sidney Kimmel Cancer Center,2 Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland,3 Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana4

Received 7 July 2004/ Accepted 9 September 2004


arrow
ABSTRACT
 
The Epstein-Barr virus (EBV) BamHI-A rightward transcripts, or BARTs, are a family of mRNAs expressed in all EBV latency programs, including EBV-infected B cells in healthy carriers. Despite their ubiquitous expression, the regulation and biological function of BARTs are still unclear. In this study, the BART 5' termini were characterized by using a procedure that selects capped, full-length mRNAs. Two TATA-less promoter regions, designated P1 and P2, were mapped. P1 had relatively high basal activity in both epithelial and B cells, whereas P2 exhibited higher activity in epithelial cells. Upon EBV infection of B cells, transcription from P1 was detected soon after infection, while expression from P2 was delayed. Promoter-reporter assays in transiently transfected cells revealed that P1 and P2 were differentially regulated. Interferon regulatory factor 7 (IRF7) and IRF5 negatively regulated P1 activity. c-Myc and C/EBP family members positively regulated P2. Regulation of P2 by C/EBPs was characterized by electrophoretic mobility shift assay, chromatin immunoprecipitation, and reporter assays. More-abundant BART expression in epithelial cells correlated with the relative expression of positive and negative regulators in these cells.


arrow
INTRODUCTION
 
The BamHI-A rightward transcripts (BARTs), also known as complementary strand transcripts, are complexly spliced Epstein-Barr virus (EBV) mRNAs with a common 3' open reading frame and polyadenylation signal. BARTs were first identified in nasopharyngeal carcinoma tissues (11, 17, 18, 20) but are expressed in all EBV-associated malignancies, including Hodgkin's disease and gastric carcinoma (54, 71). BARTs are also detected at lower levels in all latently EBV-infected B-cell lines in culture (4, 11, 25, 47, 71) and in peripheral blood B cells from healthy EBV-positive donors (10). Recently, it has been found that the BART intronic regions generate micro-RNAs, one of which potentially targets the viral DNA polymerase BALF5 transcripts for degradation (43). The BARTs also contain several open reading frames, BARF0, RK-BARF0, A73, and RPMS1 (15, 18, 25, 27, 34, 47, 52, 53). Although it has been difficult to convincingly demonstrate expression of the protein products of these open reading frames in EBV-infected cells (62), immunological data suggestive of protein expression have been obtained. Cytotoxic T cells isolated from healthy seropositive donors recognized BARF0-transfected cells (28), and serum from patients with nasopharyngeal carcinoma precipitated in vitro-translated BARF0 polypeptides (18).

Experiments performed with the exogenously expressed BART-encoded proteins RPMS1, A73, and RK-BARF0 have suggested roles in modifying Notch pathway responses and in activities orchestrated by the adaptor protein RACK1. RPMS1 interacted with CBF1 in yeast two-hybrid and glutathione S-transferase affinity assays and with the CBF1-interacting corepressor CIR in glutathione S-transferase affinity, coimmunoprecipitation, and mammalian two-hybrid assays (52, 71). CBF1 (CSL, RBP-Jk) is the nuclear effector of the Notch signaling pathway and a promoter targeting partner for EBNA2. CBF1 binds to the consensus sequence GTGGGAA (19, 37, 60) and recruits a corepressor complex that includes SMRT/NcoR, SAP30, CIR, SKIP, HDAC1/2, and MINT/SHARP (22, 24, 31, 42, 74, 75). EBNA2 and the activated form of Notch, NotchIC, convert CBF1 from a transcriptional repressor to a mediator of transcriptional activation by displacing SMRT from its contacts on CBF1 and SKIP. Binding of SMRT to SKIP and binding of SMRT to CBF1 is mutually exclusive with binding of these two proteins to EBNA2 or to NotchIC. Recruitment of basal transcription factors (56, 58) and coactivators by EBNA2 (57, 63, 65, 68) and NotchIC (16, 29, 32, 33, 64, 70) completes the transition from repression to activation. In transient-expression assays, RPMS1 interfered with EBNA2 and NotchIC activation of reporters driven by a promoter containing CBF1 binding sites and also, when constitutively expressed in C2C12 cells, partially blocked the ability of NotchIC to prevent myoblast differentiation (52, 71). RK-BARF0 was found to interact with the extracellular ligand binding domain of Notch4 in a yeast two-hybrid screen. RK-BARF0 has a potential endoplasmic reticulum targeting signal peptide sequence and may interact with Notch in the Golgi network prior to the transport of Notch to the plasma membrane. It has been suggested that the interaction of RK-BARF0 with Notch may stimulate nuclear Notch activity (34). A yeast two-hybrid screen identified RACK1 as an A73-interacting protein (52). RACK1 is an adaptor protein that binds to certain forms of protein kinase C (38), associates with the alpha/beta interferon receptor (12, 61), and is targeted by the adenovirus E1A protein (48).

Exactly how the RPMS1, A73, and RK-BARF0 proteins would contribute to EBV infection or biology is not clear. Recombinant EBV with a deletion of the region of the genome that forms the template for the BARTs is capable of immortalizing peripheral blood mononuclear cells in vitro (26, 45), implying that the BARTs are dispensable, at least in cultured B cells. To better understand the circumstances in which the BARTs may be expressed in vivo, capped BART mRNAs were isolated and the associated upstream regulatory regions were characterized.


arrow
MATERIALS AND METHODS
 
Plasmids. BART promoter reporters were constructed by PCR amplification with the following primers: HC71, 5'(150317)-GTAGCTACGGCCAAGGGCAG-3' and 5'(150539)-CTGACAGTTTAGGAATGACTCA-3'; HC82, 5'(150317)-GTAGCTACGGCCAAGGGCAG-3' and 5'(150661)-GCAGCTTGAAAAATGGCAAC-3'; HC114, 5'(149761)-GTGTTAGTACCTGTCCATTC-3' and 5'(150475)-ATTTTTCTCAGATCTGGTTAAATTTG-3'; HC116, 5'(149761)-GTGTTAGTACCTGTCCATTC-3' and 5'(150310)-CAACGGAACTCATATTAACTAAC-3'. The PCR products were ligated between the HindIII and XbaI sites of pCAT-BASIC (Promega). C/EBP{alpha}, C/EBPß, and C/EBP{delta} were PCR amplified from HeLa cDNA and cloned into a pSG5 (Stratagene) vector modified with a Flag epitope (pJH253). An interleukin 6 (IL-6) promoter fragment was PCR amplified from HeLa cell DNA with the primers 5'-GATCCTCCTGCAAGAGACAC-3' and 5'-CAGAATGAGCCTCAGAGACATC-3' and ligated into pGL2 (Amersham-Pharmacia) to generate an IL-6-luciferase reporter. c-Myc and c-Myc-mt were obtained from Chi Dang (Johns Hopkins School of Medicine). Expression vectors for interferon regulatory factor 1 (IRF1), IRF3, and IRF7 were obtained from Gary Hayward (Johns Hopkins School of Medicine) and Yan Yuan (76), and an IRF5 vector was obtained from Jae Myun Lee (Johns Hopkins School of Medicine).

Cell lines and virus preparation. The EBV-positive cell lines B95-8, IB4, Raji, Rael, Akata, Akata-Bx1, and C666-1 and the EBV-negative cell lines DG75 and Akata 4E3 were maintained in RPMI 1640 plus 10% fetal bovine serum. LCL-III was grown in RPMI plus 15% fetal bovine serum and was a gift from Elliott Kieff. HeLa cells, EBV-infected HeLa cells (HeLa-Bx1) (7), and 293-Phoenix cells were maintained in Dulbecco's minimal essential medium plus 10% fetal bovine serum. The gastric carcinoma cell line AGS (American Type Culture Collection), the EBV-converted AGS cell line AGS-Bx1 (3), the EBV-negative nasopharyngeal carcinoma cell line CNE, the immortalized normal breast epithelial cell line HBL100, and the breast cancer cell line MDA-MB468 were maintained in F-12 medium plus 10% fetal bovine serum. Cell-free EBV virus was prepared by immunoglobulin G (IgG) induction of Akata-Bx1, filtration of the cell supernatant, and concentration by centrifugation as previously described (7). EBV-negative Akata 4E3 (49) cells or peripheral blood mononuclear cells (PBMCs) were incubated with virus for 6 h, at which time the medium was replaced. Cells were harvested at different time points postinfection, and RNA was extracted by using a GenElute direct mRNA miniprep kit (Sigma).

Transient-transfection and reporter assays. HeLa cells were transfected by the calcium phosphate method, and DG75 cells were transfected by electroporation (Gene Pulser; Bio-Rad). Transfections in HeLa cells used 1 µg of reporter and 1 µg of effector DNA per 5 x 105 cells, and DG75 cells were electroporated with 10 µg of reporter and 10 µg of effector DNA per 10 x 106 cells.

RLM-RACE and RT-PCR. RNA was isolated from EBV-infected cells as previously described (7). The BART promoter region was amplified by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) by following the manufacturer's protocol (Ambion). The EBV-specific primers used were P1, 5'-GACCTGATCCTGCAGATATC-3', and P2, 5'-GCAGCTTGAAAAATGGCAAC-3'. Amplified cDNA fragments were cloned and sequenced. The primers used for reverse transcription (RT)-PCR amplification of P2-initiated products shown in Fig. 1C were 5'-CACAGCAGCACAATAGAAGCAC-3' and 5'-GACCTGATCCTGCAGATATC-3', and the primers used to clone and sequence P2-initiated products from Rael cells were 5'-CAACTCGATCCGAGAGACCG-3' or 5'-GCACAAACAGACCCCACACAG-3' and the RPMS1 3' primer 5'-CCAACGAGGCTGACCTGATC-3'. Primers for RT-PCR amplification of RPMS1- and EBNA1-spliced transcripts have been described previously (8, 71).




View larger version (38K):
[in this window]
[in a new window]
  		FIG. 1. Identification of two RNA start sites for BARTs. (A) Southern blot of cDNAs isolated from EBV-positive Rael and Akata B cells, C666-1 nasopharyngeal carcinoma cells, and NPC tissue by RLM-RACE with a primer within the RPMS1 open reading frame. Major and minor products (arrows) were detected by using an RPMS1 open reading frame oligonucleotide probe. (B and C) Ethidium bromide-stained gels showing the products of reactions for P2-initiated RNAs. (B) Product generated by RLM-RACE. (C) Upper panel, products generated by RT-PCR for P2 in different cell lines; lower panel, control RT-PCR for the RPMS1 open reading frame. (D) Structure of individual clones of the RLM-RACE (a to c) and RT-PCR (d and e) cDNAs obtained from Rael cells. Open reading frames are indicated by open boxes and numbered (I to V) according to the method of Smith et al. (52).

EMSA. Electrophoretic mobility shift assays (EMSAs) were performed as described previously (21). Briefly, double-stranded probes for two putative C/EBP binding sites in the P2 promoter were prepared with the oligonucleotides 5'-GTAACCTGCGCAAGGGTCACATTG-3' (site 1, sense strand) and 5'-GAGTCATTTACCCATTCTAGGGTAAG-3' (site 2, sense strand). A 189-bp DNA fragment covering both P2 binding sites was prepared by PCR amplification with the primers 5'-GGCCACACTGCAATTTCTCAG-3' and 5'-CAACTGCCCTTGGCCGTAG-3'. Probes were labeled with [32P]dCTP by using Klenow polymerase. For the binding assays, Flag-tagged C/EBP proteins were in vitro transcribed and translated by using the quick-coupled transcription translation system (Promega). Supershifts were performed with anti-Flag antibody (Sigma).

ChIP. HeLa-Bx1 cells were transfected with Flag-C/EBPß (HC108B) or Flag-IRF7, harvested 40 h after transfection, and lysed by using a chromatin immunoprecipitation (ChIP) kit (Upstate). Anti-Flag or anti-C/EBPß antibody or normal human IgG (5 to 10 µg per reaction) were used in the precipitation reactions. PCR detection used primers specific for the P2 promoter, the P1 promoter, or the BBLF4 open reading frame. The P1 primers amplified a 243-bp fragment between nucleotides 150361 and 150704. The BBLF4 primers amplified a 218-bp fragment between nucleotides 112908 and 112680.


arrow
RESULTS
 
Analysis of 5' termini of BARTs. BARTs are a family of alternatively spliced transcripts that are 3' coterminal (25, 47, 53). Two potential 5' termini have been identified by RT-PCR and cDNA isolation (47, 52, 53). Functional analysis of the proposed promoter region suggested that an AP-1 site and a region termed site B contributed positively to expression (14). To further define the 5' termini, we used a modified RACE protocol, RLM-RACE, in which the 3' primer was derived from exon V in the RPMS1 open reading frame (52) and the 5' primer is specific for 5'-capped mRNAs. This protocol is designed to specifically amplify full-length mRNAs. RNA is treated with calf intestinal phosphatase, which degrades uncapped RNA. The cap is then removed, and an adapter oligonucleotide is ligated to the 5' terminus. The 5' primers for PCR amplification match the adaptor oligonucleotide. Using this protocol, a major product and a minor product were obtained from EBV-positive B-cell lines (Rael and Akata), from a nasopharyngeal carcinoma cell line (C666-1), and from nasopharyngeal carcinoma biopsy tissue (NPC) (Fig. 1A). The major RACE product was isolated, cloned, and sequenced. The sequences obtained identified an RNA start site at genomic position 150641 and also indicated that differential splicing occurs within the 5' region of BARTs (Fig. 1D, a and b).

To determine whether the longer RNA product represented a second RNA start site, the RLM-RACE protocol was repeated in Rael cells with a 3' primer located 60 bp downstream of the P1 initiation position. A specific product was obtained (Fig. 1B), and cloning and sequencing of this product identified the P2 RNA initiation site at position 150357 (Fig. 1D, c). To further examine the prevalence of P2-initiated transcripts, RT-PCR was performed with a 5' primer specific for P2 and a 3' primer from the RPMS1 open reading frame. P2-initiated products were detected in all cell lines tested except Raji (Fig. 1C). RT-PCR for the RPMS1 open reading frame was used as a control (Fig. 1C, lower panel). It is interesting that IB4, which is infected with B95-8 virus, does not express the intact RPMS1 open reading frame because of the B95-8 deletion but does still express P2-initiated BARTs. The P2-specific RT-PCR products obtained from Rael cells with two different 5' primers were isolated, cloned, and sequenced. The sequences obtained also provided evidence for alternative splicing in the 5' region of P2-initiated BARTs (Fig. 1D, d and e). This is likely to account for the differently sized P2-initiated products amplified by RT-PCR from the different cell lines. The variations in 5' splicing also mean that the size of the RT-PCR products is not definitive in identifying P1- or P2-initiated mRNAs.

Expression of BARTs during EBV infection. The existence of two RNA initiation sites for the BARTs in latently infected cell lines and NPC tissue raised the possibility that the P1 and P2 transcripts may be differentially regulated. The expression of P1- and P2-initiated BARTs was therefore examined during EBV infection of PBMCs. Akata-Bx1 virus was used to infect PBMCs, and EBV latency gene expression was monitored by RT-PCR (Fig. 2A). BART RNA initiating from the P1 start site was detected 18 h after infection. This was later than the time (6 h) at which EBNA-LP was first detected but prior to the detection of EBNA1 transcripts. In contrast, P2-initiated RNA was not detected during the first 48 h after infection of PBMC. The expression of P2-initiated transcripts was also examined after EBV infection of EBV-negative Akata 4E3 cells (Fig. 2B). In this setting, expression of P2-initiated BARTs occurred 24 h postinfection at the same time as EBNA1 transcripts became detectable. Overall BART expression, as measured by detection of RPMS1-spliced RNA, again initiated at a much earlier time point (6 h). These results suggest that the P1 promoter is constitutively active in B cells, allowing BARTs to be expressed very early after EBV infection, while the P2 promoter is regulated and its activity is dependent on the induction of cellular or viral factors.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2. BART promoter usage after EBV infection of B cells. Ethidium bromide-stained gels showing the products of RT-PCRs to detect EBV transcripts present at the indicated hours postinfection with Akata Bx1 virus. (A) Infection of PBMC. ctr, positive control RT-PCR from C666-1 cells. (B) Infection of EBV-negative Akata 4E3 cells.

Verification of functional P1 and P2 promoter activity in reporter assays. The sequences surrounding the P1 and P2 RNA initiation sites are presented in Fig. 3A. No obvious TATA box sequences are discernible upstream of either initiation site, implying that the BARTs are expressed from TATA-less promoters. Potential binding sites for a variety of transcription factors are present in the P1 and P2 upstream sequences (Fig. 3A). P1-chloramphenicol acetyltransferase (CAT) (HC82) and P2-CAT (HC114) reporters were constructed along with CAT reporters carrying P1 and P2 upstream sequences but lacking the P1 and P2 sites and downstream sequences (HC71 and HC116) (Fig. 3B). CAT activity was detected in HeLa and DG75 cells with the P1 and P2 reporters, but the 3' deletion reporters were not active (Fig. 3B). P2 was expressed more actively in HeLa cells than in DG75 cells.




View larger version (40K):
[in this window]
[in a new window]
  		FIG. 3. BART promoter regions and basal activity. (A) Sequence of the 5' promoter regions showing the locations of putative transcription factor binding sites (boldface type, underlined), P1 and P2 mRNA initiation sites (arrowheads), 5' splice donor sites (arrows), and the 3' termini of the reporter constructs shown in panel B (hearts). Site B is a transcription factor binding site determined by in vivo footprinting (14). 150061 is the sequence number from the EBV B95-8 genome. (B) Structure of P1 and P2 reporter plasmids. The basal activity of these plasmids in HeLa and DG75 B cells is given relative to that of a Qp-CAT reporter (set at +++).

The P1 promoter is downregulated by IRFs. The P1 promoter region contains potential AP-1 and IRF sites (Fig. 3A). Cotransfection of c-Jun with P1-CAT into DG75 cells resulted in only a small increase in reporter expression, perhaps because c-Jun is not limiting in these cells (Fig. 4). However, cotransfection of a nonfunctional mutant c-Jun resulted in a fourfold reduction, suggesting that c-Jun or Jun family members do contribute to P1 activity. Cotransfection of P1-CAT into HeLa cells with IRF expression plasmids showed that P1 is negatively regulated by IRFs (Fig. 5A). Cotransfection of IRF1 had a small negative effect, while P1 was downregulated 4.7-fold by IRF5 and 6.5-fold by IRF7. Downregulation of endogenous BART expression by IRF7 was confirmed in EBV-converted HeLa-Bx1 cells (Fig. 5B). Transfection of an IRF7 expression vector into HeLa-Bx1 cells resulted in a downregulation of expression of the RPMS1 open reading frame containing BARTs that was measured as a fivefold reduction by quantitative real-time RT-PCR. IRF7 is known to repress expression from Qp, and Qp-initiated transcripts were found to be downregulated sevenfold in the same experiment. Further, a ChIP assay performed on Flag-IRF-7-transfected HeLa-Bx1 cells showed binding of Flag-IRF7 to the endogenous BART promoter (Fig. 5C). P2 promoter DNA was seen associated with the anti-Flag precipitate when P2-specific primers were used but not when primers for the BBLF4 open reading frame were used. No PCR product was detected in immunoprecipitates formed with control antibody.



View larger version (8K):
[in this window]
[in a new window]
  		FIG. 4. Regulation of the P1 promoter by cJun. Reporter assay in which P1-CAT was cotransfected into DG75 cells with vector (bar 1), expression vectors for c-Jun (bar 2), or a c-Jun mutant (bar 3).



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5. Regulation of the P1 promoter by IRFs. (A) Reporter assay in which P1-CAT was cotransfected into HeLa cells with vector (v) or with expression plasmids for IRF1, IRF5, or IRF7. The assays were performed three times, and standard deviations are indicated. (B) Ethidium bromide-stained gel showing the products of RT-PCR for RPMS1 and Qp-EBNA1 in HeLa-Bx1 cells transfected with vector or with an expression vector for IRF7. Cellular TATA-binding protein served as a control. The fold-down regulation measured by real-time RT-PCR on the same samples is given in parentheses. (C) ChIP assay showing association of the endogenous P1 promoter in HeLa-Bx1 cells with IRF7. PCRs for the BBLF4 open reading frame (ORF) and immunoprecipitation with nonspecific antibody were used as negative controls. {alpha}Flag, anti-Flag.

The P2 promoter is positively regulated by c-Myc and C/EBP proteins. Potential binding sites for c-Myc and C/EBP are present in the P2 promoter. A reporter assay performed in transfected HeLa cells showed a dose-dependent activation of P2-CAT by cotransfected wild-type c-Myc and no effect of a control mutant c-Myc (Fig. 6). C/EBPs are a family of bZIP transcription factors that bind to the same consensus DNA motif in vitro and regulate proliferation and differentiation responses. Cotransfection of P2-CAT with C/EBPß or C/EBP{delta} strongly activated P2-driven expression in both HeLa (Fig. 7A) and DG75 (Fig. 7B) cells, and activation by C/EBP{alpha} was also shown in DG75 cells (Fig. 7B). The response of the P2 promoter to C/EBPß was comparable to that seen with the promoter for IL-6, which is known to be regulated by C/EBPß (Fig. 7C).



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 6. P2 is upregulated by c-Myc. Reporter assay in which P2-CAT was cotransfected into HeLa cells with vector (v) or expression vectors for c-Myc (wt) or a mutant c-Myc (mt) and CAT activity was measured. The assay was performed three times, and standard deviations are indicated.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 7. P2 is positively regulated by C/EBP family proteins. Reporter assays showing the response of P2-CAT to cotransfection with vector (v), C/EBPß, or C/EBP{delta} in HeLa cells (A) and C/EBP{alpha}, C/EBPß, or C/EBP{delta} in DG75 B cells (B). (C) Luciferase assay showing the response of a control IL-6-luciferase reporter to cotransfection with vector (v) or C/EBPß in HeLa cells. The assay was performed three times, and standard deviations are indicated.

The strong response of P2 to C/EBP proteins suggested that these proteins may be key regulators of BART expression, and the interaction of the P2 promoter with C/EBP proteins was further characterized. C/EBP binding sites can vary quite widely in sequence, and functionality is difficult to predict. To confirm that the putative C/EBP sites in P2 were capable of binding C/EBPs, EMSAs were performed with in vitro-translated Flag-tagged C/EBP{alpha}, C/EBPß, and C/EBP{delta} proteins and 32P-labeled oligonucleotide probes representing the two putative C/EBP sites in P2. All three Flag-tagged C/EBP proteins bound to each site (Fig. 8A and B). The identity of the shifted species was confirmed with anti-Flag antibody, which generated a supershifted band in each case. The ability of both C/EBP sites to be occupied concurrently was tested by using as probe a P2 DNA fragment that spanned both C/EBP binding sites and in vitro-translated Flag-C/EBP{alpha}. In the EMSA (Fig. 8C), a strong band and a second weak band were seen upon addition of Flag-C/EBP{alpha}. Two bands were more readily apparent in the supershift generated by the addition of anti-Flag antibody. This latter result suggests that both C/EBP sites on the P2 promoter can be occupied at the same time.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 8. P2 promoter contains functional C/EBP binding sites. (A, B) EMSAs showing binding of in vitro-translated, Flag-tagged C/EBP{alpha}, C/EBPß, and C/EBP{delta} to 32P-labeled oligonucleotide probes representing the two C/EBP sites identified in the P2 promoter sequence. SS, supershifted band. (A) Site 1; (B) site 2. (C) EMSA with a 160-bp DNA fragment probe from P2 that covers both C/EBP binding sites and in vitro-translated, Flag-tagged C/EBP{alpha}. The supershift (SS) shows two shifted species. {alpha}-Flag, anti-Flag; {alpha}-C/EBP{alpha}, anti-C/EBP{alpha}; +, present; –, absent.

Regulation of P2 by C/EBPß in epithelial cells. The expression of BARTs is more abundant in EBV-associated epithelial malignancies than in EBV-positive B-cell lines (18). To determine whether there was any relationship between BART expression and that of the early response C/EBP family member C/EBPß, a Western blot was performed to examine C/EBPß protein expression in a variety of B-cell lines and epithelial cell lines (Fig. 9). Three forms of C/EBPß protein are expressed by using alternative initiator methionines. LAP1 and LAP2 are transcriptional activators, and the small LIP form consists of the dimerization and DNA binding domains and is a negative modulator of LAP activity. An extract from 293 cells transfected with Flag-C/EBPß was included in the analysis to provide a marker for these different forms. The tumor-derived epithelial cell lines CNE (nasopharyngeal carcinoma), MB468 (breast cancer), and HeLa (cervical cancer) expressed C/EBPß at readily detectable levels. The in vitro-immortalized normal epithelial cell line HBL100 and the Burkitt lymphoma-derived DG75 and Akata B-cell lines had barely detectable levels of C/EBPß, although Akata cells did express the negative LIP form of C/EBPß. Expression of C/EBPß in the in vitro-immortalized B-cell lines IB4 and LCL(III) was below the level of detection. Thus, C/EBPß expression is significantly higher in epithelial tumor cell lines than in tumor-derived B-cell lines and higher in cell lines derived from normal epithelium than in cell lines derived from normal B cells.



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 9. Differential expression of C/EBPß in epithelial and B-cell lines. A Western blot probed with C/EBPß antibody (upper) and control ß-actin antibody (lower) is shown. The C/EBPß lane contained extract from C/EBPß-transfected 293 cells. LAP1, LAP2, and LIP are different forms of C/EBPß generated from alternative translational start sites.

Additional evidence for regulation of BARTs by C/EBPß was obtained by examining the endogenous promoter in EBV-converted HeLa-Bx1 cells, a ChIP assay performed on HeLa-Bx1 cells transfected with Flag-tagged C/EBPß found association of C/EBPß with the P2 promoter region when the immunoprecipitations were performed with either anti-C/EBPß antibody or anti-Flag antibody. Control antibody and control PCRs were appropriately negative (Fig. 10A). BART expression, as measured by expression of the RPMS1 open reading frame, was increased fourfold in HeLa-Bx1 cells transfected with C/EBPß (Fig. 10B) Expression of the endogenous C/EBPß-responsive cellular IL-6 gene was also measured and showed a sixfold increase in the C/EBPß-transfected HeLa-Bx1 cells. Quantitation of RNA expression was obtained by using real-time RT-PCR. Since the transfection efficiency is less than 100%, the relative induction by C/EBPß is underestimated in this assay.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 10. C/EBPß binds to the P2 promoter and induces BART expression in HeLa-Bx1 cells. (A) ChIP assay showing PCR amplification of P2 promoter fragments from Flag-C/EBPß-transfected HeLa-Bx1 cells immunoprecipitated with anti-C/EBPß ({alpha}-C/EBPß) and anti-Flag ({alpha}-Flag) antibodies but not with control antibody (Ab) against human IgG. DNA(–), no DNA added to the PCR. (B) Ethidium bromide-stained products from RT-PCR amplification of EBV RPMS1 and cellular IL-6 mRNAs in HeLa-Bx1 cells transfected with empty vector or a C/EBPß expression vector. The induction determined by real-time RT-PCR analysis of the same samples is given in parentheses.


arrow
DISCUSSION
 
Amplification of capped mRNA for the BARTs led to the identification of two RNA start sites, P1 (150648) and P2 (150343). P1 is the same as the start site for the 22.2 BART transcript (150640) (52, 53) and C15 5' RACE clones (47), and P2 is likely to represent the initiation site for the transcripts described with 5' endpoints at 150519 (47). Examination of expression of the P1- and P2-associated TATA-less promoter regions by using P1 and P2 reporters along with analysis by RT-PCR of expression of the endogenous P1- and P2-regulated transcripts provided supporting evidence for the existence of two differentially regulated promoter regions. P1 is expressed soon after EBV infection of B cells at around 6 h postinfection, while expression from P2 occurs days after infection, as opposed to hours. This use of two regulatory regions for BART expression is reminiscent of the Wp to Cp switch that occurs after B-cell infection and allows a conversion from cell-regulated (Wp) to virally modulated (Cp) expression of the EBNA latency genes (66, 67). Having two control regions also allows tissue-specific or signal transduction-specific expression after establishment of infection. LMP1 is also expressed from two promoters that are responsive to completely different transcription factors (6, 9, 23, 35, 46, 50, 51, 59).

As has been noted previously (14), the P1 region contains potential SP1 and AP-1 sites that could contribute to constitutive P1 expression. The P1 promoter responded only minimally to transfected cJun in our experiments with DG75 cells, but there was a fourfold downregulation in cells cotransfected with a c-Jun mutant, suggesting that this family of bZip proteins does contribute to P1 activity. Downregulation of P1 appears to be brought about by IRF family members, particularly IRF5 and IRF7. Comparatively little is known about IRF5 function. IRF5 is constitutively expressed at higher levels in B cells and dendritic cells than in other cell types and is activated by certain viruses. IRF5 participates in alpha interferon responses and p53 induction (1, 2, 39). Downregulation by IRF7 is interesting in that IRF7 is activated and rendered nuclear by LMP1 (72). LMP1 expression in PBMCs in culture is delayed and is detected 2 to 3 days after EBV infection, a time that would coincide with the onset of P2 usage. In contrast to the situation with the Wp and Cp EBNA promoters, where Wp is generally extinguished by methylation (36) and Cp becomes the sole promoter driving the EBNAs in latency III (55), the region encompassing both P1 and P2 promoters is hypomethylated, at least in C15 NPC tumor-associated virus (14).

The complexity of the BART splicing patterns is reemphasized by the presence of splicing variations in the 5' region of the P2 initiated cDNAs. These splicing variations preclude using the size of the RACE products to definitively identify P1- or P2-initiated transcripts. However, it appears that P1 is the dominant start site for BARTs containing the RPMS1 open reading frame in the Rael and Akata cell lines, NPC tumor tissue, and the C666-1 cell line. Rael and Akata are B cells that have a type I latency phenotype, do not express LMP1, and express relatively low levels of IRF7 (41). NPC tumors are heterogeneous for LMP1 expression, but IRF7 expression is not constitutive in this cell background. The predominant use of P1 for RPMS1-containing BARTs in these cells is thus consistent with a lack of IRF7-mediated downregulation. Expression of BARTs in the absence of detectable P2 usage in LMP1-expressing Raji B cells may imply that regulation of P1 by IRFs is more complex than would be predicted by a model of LMP1-mediated IRF7 activation. A similar situation exists for the EBNA1 Qp promoter, which is negatively regulated by IRF7 (73) but is nonetheless utilized to drive EBNA1 in settings of type II latency, such as Hodgkin's disease (13).

The P2 promoter was upregulated in reporter assays by c-Myc and the C/EBP family members C/EBP{alpha}, C/EBPß, and C/EBP{delta}. LMP1 positively regulates c-Myc expression through IL-6 (7), and c-Myc is consistently upregulated in Burkitt lymphoma cells as a consequence of chromosomal translocation (30). Only a few studies have examined the contribution of C/EBP proteins to EBV gene expression. C/EBP{delta} has been described as a regulator of the latency Cp (40). C/EBP{alpha} expression is induced by treatment of Akata cells with anti-IgG antibody, and C/EBP{alpha} stimulates basal expression of the lytic BZLF1 promoter and also cooperates with Zta in positive autoregulation of Zta expression (69). Regulation of P2 by C/EBP{alpha} would potentially result in expression of the BARTs during lytic induction.

The endogenous P2 promoter in HeLa-Bx1 cells was upregulated by transfected C/EBPß to a similar extent as a known C/EBPß responsive gene, cellular IL-6, and association of C/EBPß with the endogenous BART promoter was also shown by ChIP analysis. The positive response of the P2 promoter to C/EBPß may contribute to the higher levels of expression of the BARTs reported in epithelial cells than in B cells. C/EBPß is an acute-phase response gene that is not constitutively expressed in the B-cell compartment (44). Western blot examination of C/EBPß protein levels in B-cell lines revealed low levels of the LAP and LIP forms in EBV-negative DG75 Burkitt lymphoma cells, significant levels of only the negative regulatory LIP form in Akata Burkitt lymphoma cells, and undetectable levels of all forms in the in vitro-immortalized LCLs IB4 and LCL (III). The in vitro-immortalized HBL100 cell line that is derived from normal breast epithelium also showed only low levels of LAP and LIP. In contrast, C/EBPß forms were expressed at significantly higher levels in cancer-derived epithelial cell lines. LAP2 was readily detectable in the HeLa cervical cancer cell line, as was LAP2 and LIP in the MB468 breast cancer cell line. LAP2 is frequently upregulated in breast carcinoma tissue, and introduction of LAP2 into normal breast epithelial cells is sufficient to lead to anchorage-independent growth and formation of foci (5). Strong expression of all three C/EBPß forms was also observed in the EBV-negative nasopharyngeal carcinoma-derived CNE cells, a relevant cell line for EBV biology.

In summary, characterization of the TATA-less BART promoter strengthened evidence for the existence of two regulatory regions that are utilized at different times after primary infection. The positive regulation by C/EBP family members is likely to contribute to the higher expression of BARTs seen in EBV-infected epithelial cells.


arrow
ACKNOWLEDGMENTS
 
We thank D. Huang for C666-1 cells, M. Ng for NPC tissue, and Feng Chang for manuscript preparation.

This work was funded by Public Health Services grants RO1 CA30356 to S.D.H. and R01 AI20662 to L.H.-F.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Johns Hopkins University School of Medicine, Bunting-Blaustein Building CRB308, 1650 Orleans St., Baltimore, MD 21231-1000. Phone: (410) 614-0592. Fax: (410) 502-6802. E-mail: dhayward{at}jhmi.edu. Back


arrow
REFERENCES
 
    1
  1. Barnes, B. J., A. E. Field, and P. M. Pitha-Rowe. 2003. Virus-induced heterodimer formation between IRF-5 and IRF-7 modulates assembly of the IFNA enhanceosome in vivo and transcriptional activity of IFNA genes. J. Biol. Chem. 278:16630-16641.[Abstract/Free Full Text]
    2. 2
  2. Barnes, B. J., M. J. Kellum, K. E. Pinder, J. A. Frisancho, and P. M. Pitha. 2003. Interferon regulatory factor 5, a novel mediator of cell cycle arrest and cell death. Cancer Res. 63:6424-6431.[Abstract/Free Full Text]
    3. 3
  3. Borza, C. M., and L. M. Hutt-Fletcher. 2002. Alternate replication in B cells and epithelial cells switches tropism of Epstein-Barr virus. Nat. Med. 8:594-599.[CrossRef][Medline]
    4. 4
  4. Brooks, L. A., A. L. Lear, L. S. Young, and A. B. Rickinson. 1993. Transcripts from the Epstein-Barr virus BamHI A fragment are detectable in all three forms of virus latency. J. Virol. 67:3182-3190.[Abstract/Free Full Text]
    5. 5
  5. Bundy, L. M., and L. Sealy. 2003. CCAAT/enhancer binding protein beta (C/EBPbeta)-2 transforms normal mammary epithelial cells and induces epithelial to mesenchymal transition in culture. Oncogene 22:869-883.[CrossRef][Medline]
    6. 6
  6. Chang, M. H., C. K. Ng, Y. J. Lin, C. L. Liang, P. J. Chung, M. L. Chen, Y. S. Tyan, C. Y. Hsu, C. H. Shu, and Y. S. Chang. 1997. Identification of a promoter for the latent membrane protein 1 gene of Epstein-Barr virus that is specifically activated in human epithelial cells. DNA Cell Biol. 16:829-837.[Medline]
    7. 7
  7. Chen, H., L. Hutt-Fletcher, L. Cao, and S. D. Hayward. 2003. A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus. J. Virol. 77:4139-4148.[Abstract/Free Full Text]
    8. 8
  8. Chen, H., J. M. Lee, Y. Wang, D. P. Huang, R. F. Ambinder, and S. D. Hayward. 1999. The Epstein-Barr virus latency Qp promoter is positively regulated by STATs and Zta interference with JAK-STAT activation leads to loss of Qp activity. Proc. Natl. Acad. Sci. USA 96:9339-9344.[Abstract/Free Full Text]
    9. 9
  9. Chen, H., J. M. Lee, Y. Zong, M. Borowitz, M. H. Ng, R. F. Ambinder, and S. D. Hayward. 2001. Linkage between STAT regulation and Epstein-Barr virus gene expression in tumors. J. Virol. 75:2929-2937.[Abstract/Free Full Text]
    10. 10
  10. Chen, H., P. Smith, R. F. Ambinder, and S. D. Hayward. 1999. Expression of Epstein-Barr virus BamHI-A rightward transcripts (BARTs) in latently infected B cells from peripheral blood. Blood 93:3026-3032.[Abstract/Free Full Text]
    11. 11
  11. Chen, H.-L., M. M. L. Lung, J. S. T. Sham, D. T. K. Choy, B. E. Griffin, and M. H. Ng. 1992. Transcription of BamHI-A region of the EBV genome in NPC tissues and B cells. Virology 191:193-201.[CrossRef][Medline]
    12. 12
  12. Croze, E., A. Usacheva, D. Asarnow, R. D. Minshall, H. D. Perez, and O. Colamonici. 2000. Receptor for activated C-kinase (RACK-1), a WD motif-containing protein, specifically associates with the human type I IFN receptor. J. Immunol. 165:5127-5132.[Abstract/Free Full Text]
    13. 13
  13. Deacon, E. M., G. Pallesen, G. Niedobitek, J. Crocker, L. Brooks, A. B. Rickinson, and L. S. Young. 1993. Epstein-Barr virus and Hodgkin's disease: transcriptional analysis of virus latency in the malignant cells. J. Exp. Med. 177:339-349.[Abstract/Free Full Text]
    14. 14
  14. de Jesus, O., P. R. Smith, L. C. Spender, C. Elgueta Karstegl, H. H. Niller, D. Huang, and P. J. Farrell. 2003. Updated Epstein-Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs of EBV. J. Gen. Virol. 84:1443-1450.[Abstract/Free Full Text]
    15. 15
  15. Fries, K. L., T. B. Sculley, J. Webster-Cyriaque, P. Rajadurai, R. H. Sadler, and N. Raab-Traub. 1997. Identification of a novel protein encoded by the BamHI A region of the Epstein-Barr virus. J. Virol. 71:2765-2771.[Abstract]
    16. 16
  16. Fryer, C. J., E. Lamar, I. Turbachova, C. Kintner, and K. A. Jones. 2002. Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex. Genes Dev. 16:1397-1411.[Abstract/Free Full Text]
    17. 17
  17. Gilligan, K., H. Sato, P. Rajadural, P. Busson, L. Young, A. Rickinson, T. Tursz, and N. Raab-Traub. 1990. Novel transcription from the Epstein-Barr virus terminal EcoRI fragment, DIJhet, in a nasopharyngeal carcinoma. J. Virol. 64:4948-4956.[Abstract/Free Full Text]
    18. 18
  18. Gilligan, K. J., P. Rajadurai, J. C. Lin, P. Busson, M. Abdel-Hamid, U. Prasad, T. Tursz, and N. Raab-Traub. 1991. Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for viral protein expressed in vivo. J. Virol. 65:6252-6259.[Abstract/Free Full Text]
    19. 19
  19. Grossman, S. R., E. Johannsen, X. Tong, R. Yalamanchili, and E. Kieff. 1994. The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J {kappa} recombination signal binding protein. Proc. Natl. Acad. Sci. USA 91:7568-7572.[Abstract/Free Full Text]
    20. 20
  20. Hitt, M. M., M. Allday, T. Hara, L. Karran, M. D. Jones, P. Busson, T. Tursz, I. Ernberg, and B. Griffin. 1989. EBV gene expression in an NPC-related tumor. EMBO J. 8:2639-2651.[Medline]
    21. 21
  21. Hsieh, J. J.-D., T. Henkel, P. Salmon, E. Robey, M. G. Peterson, and S. D. Hayward. 1996. Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol. Cell. Biol. 16:952-959.[Abstract]
    22. 22
  22. Hsieh, J. J.-D., S. Zhou, L. Chen, D. B. Young, and S. D. Hayward. 1999. CIR, a corepressor linking the DNA binding factor CBF1 to the histone deacetylase complex. Proc. Natl. Acad. Sci. USA 96:23-28.[Abstract/Free Full Text]
    23. 23
  23. Johannsen, E., E. Koh, G. Mosialos, X. Tong, E. Kieff, and S. R. Grossman. 1995. Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J{kappa} and PU.1. J. Virol. 69:253-262.[Abstract]
    24. 24
  24. Kao, H.-Y., P. Ordentlich, N. Koyano-Nakagawa, Z. Tang, M. Downes, C. R. Kintner, R. M. Evans, and T. Kadesch. 1998. A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12:2269-2277.[Abstract/Free Full Text]
    25. 25
  25. Karran, L., Y. Gao, P. R. Smith, and B. E. Griffin. 1992. Expression of a family of complementary-strand transcripts in Epstein-Barr virus-infected cells. Proc. Natl. Acad. Sci. USA 89:8058-8062.[Abstract/Free Full Text]
    26. 26
  26. Kempkes, B., D. Pich, R. Zeidler, B. Sugden, and W. Hammerschmidt. 1995. Immortalization of human B lymphocytes by a plasmid containing 71 kilobase pairs of Epstein-Barr virus DNA. J. Virol. 69:231-238.[Abstract]
    27. 27
  27. Kienzle, N., M. Buck, S. Greco, K. Krauer, and T. B. Sculley. 1999. Epstein-Barr virus-encoded RK-BARF0 protein expression. J. Virol. 73:8902-8906.[Abstract/Free Full Text]
    28. 28
  28. Kienzle, N., T. B. Sculley, L. Poulsen, M. Buck, S. Cross, N. Raab-Traub, and R. Khanna. 1998. Identification of a cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression. J. Virol. 72:6614-6620.[Abstract/Free Full Text]
    29. 29
  29. Kitagawa, M., T. Oyama, T. Kawashima, B. Yedvobnick, A. Kumar, K. Matsuno, and K. Harigaya. 2001. A human protein with sequence similarity to Drosophila mastermind coordinates the nuclear form of notch and a CSL protein to build a transcriptional activator complex on target promoters. Mol. Cell. Biol. 21:4337-4346.[Abstract/Free Full Text]
    30. 30
  30. Klein, G. 1983. Specific chromosomal translocations and the genesis of B-cell-derived tumors in mice and men. Cell 32:311-315.[CrossRef][Medline]
    31. 31
  31. Kuroda, K., H. Han, S. Tani, K. Tanigaki, T. Tun, T. Furukawa, Y. Taniguchi, H. Kurooka, Y. Hamada, S. Toyokuni, and T. Honjo. 2003. Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway. Immunity 18:301-312.[CrossRef][Medline]
    32. 32
  32. Kurooka, H., and T. Honjo. 2000. Functional interaction between the mouse notch1 intracellular region and histone acetyltransferases PCAF and GCN5. J. Biol. Chem. 275:17211-17220.[Abstract/Free Full Text]
    33. 33
  33. Kurooka, H., K. Kuroda, and T. Honjo. 1998. Roles of the ankyrin repeats and C-terminal region of the mouse notch1 intracellular region. Nucleic Acids Res. 26:5448-5455.[Abstract/Free Full Text]
    34. 34
  34. Kusano, S., and N. Raab-Traub. 2001. An Epstein-Barr virus protein interacts with Notch. J. Virol. 75:384-395.[Abstract/Free Full Text]
    35. 35
  35. Laux, G., B. Adam, L. J. Strobl, and F. Moreau-Gachelin. 1994. The Spi-1/PU.1 and Spi-B ets family transcription factors and the recombination signal binding protein RBP-J.kappa interact with an Epstein-Barr virus nuclear antigen 2 responsive cis-element. EMBO J. 13:5624-5632.[Medline]
    36. 36
  36. Li, H., and J. Minarovits. 2003. Host cell-dependent expression of latent Epstein-Barr virus genomes: regulation by DNA methylation. Adv. Cancer Res. 89:133-156.[Medline]
    37. 37
  37. Ling, P. D., J. J.-D. Hsieh, I. K. Ruf, D. R. Rawlins, and S. D. Hayward. 1994. EBNA-2 upregulation of Epstein-Barr virus latency promoters and the cellular CD23 promoter utilizes a common targeting intermediate, CBF1. J. Virol. 68:5375-5383.[Abstract/Free Full Text]
    38. 38
  38. Mochly-Rosen, D. 1995. Localization of protein kinases by anchoring proteins: a theme in signal transduction. Science 268:247-251.[Abstract/Free Full Text]
    39. 39
  39. Mori, T., Y. Anazawa, M. Iiizumi, S. Fukuda, Y. Nakamura, and H. Arakawa. 2002. Identification of the interferon regulatory factor 5 gene (IRF-5) as a direct target for p53. Oncogene 21:2914-2918.[CrossRef][Medline]
    40. 40
  40. Nilsson, T., H. Zetterberg, Y. C. Wang, and L. Rymo. 2001. Promoter-proximal regulatory elements involved in oriP-EBNA1-independent and -dependent activation of the Epstein-Barr virus C promoter in B-lymphoid cell lines. J. Virol. 75:5796-5811.[Abstract/Free Full Text]
    41. 41
  41. Nonkwelo, C., I. K. Ruf, and J. Sample. 1997. Interferon-independent and -induced regulation of Epstein-Barr virus EBNA-1 gene transcription in Burkitt lymphoma. J. Virol. 71:6887-6897.[Abstract]
    42. 42
  42. Oswald, F., U. Kostezka, K. Astrahantseff, S. Bourteele, K. Dillinger, U. Zechner, L. Ludwig, M. Wilda, H. Hameister, W. Knochel, S. Liptay, and R. M. Schmid. 2002. SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. EMBO J. 21:5417-5426.[CrossRef][Medline]
    43. 43
  43. Pfeffer, S., M. Zavolan, F. A. Grasser, M. Chien, J. J. Russo, J. Ju, B. John, A. J. Enright, D. Marks, C. Sander, and T. Tuschl. 2004. Identification of virus-encoded microRNAs. Science 304:734-736.[Abstract/Free Full Text]
    44. 44
  44. Ramji, D. P., and P. Foka. 2002. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem. J. 365:561-575.[Medline]
    45. 45
  45. Robertson, E., and E. Kieff. 1995. Reducing the complexity of the transforming Epstein-Barr virus genome to 64 kilobase pairs. J. Virol. 69:983-993.[Abstract]
    46. 46
  46. Sadler, R. H., and N. Raab-Traub. 1995. The Epstein-Barr virus 3.5-kilobase latent membrane protein 1 mRNA initiates from a TATA-less promoter within the first terminal repeat. J. Virol. 69:4577-4581.[Abstract]
    47. 47
  47. Sadler, R. H., and N. Raab-Traub. 1995. Structural analyses of the Epstein-Barr virus BamHI A transcripts. J. Virol. 69:1132-1141.[Abstract]
    48. 48
  48. Severino, A., A. Baldi, G. Cottone, M. Han, N. Sang, A. Giordano, A. M. Mileo, M. G. Paggi, and A. De Luca. 2004. RACK1 is a functional target of the E1A oncoprotein. J. Cell. Physiol. 199:134-139.[CrossRef][Medline]
    49. 49
  49. Shimizu, N., A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada. 1994. Isolation of Epstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent on EBV. J. Virol. 68:6069-6073.[Abstract/Free Full Text]
    50. 50
  50. Sjoblom, A., A. Jansson, W. Yang, S. Lain, T. Nilsson, and L. Rymo. 1995. PU box-binding transcription factors and a POU domain protein cooperate in the Epstein-Barr virus (EBV) nuclear antigen 2-induced transactivation of the EBV latent membrane protein 1 promoter. J. Gen. Virol. 76:2679-2692.[Abstract/Free Full Text]
    51. 51
  51. Sjoblom, A., W. Yang, L. Palmqvist, A. Jansson, and L. Rymo. 1998. An ATF/CRE element mediates both EBNA2-dependent and EBNA2-independent activation of the Epstein-Barr virus LMP1 gene promoter. J. Virol. 72:1365-1376.[Abstract/Free Full Text]
    52. 52
  52. Smith, P. R., O. de Jesus, D. Turner, M. Hollyoake, C. E. Karstegl, B. E. Griffin, L. Karran, Y. Wang, S. D. Hayward, and P. J. Farrell. 2000. Structure and coding content of CST (BART) family RNAs of Epstein-Barr virus. J. Virol. 74:3082-3092.[Abstract/Free Full Text]
    53. 53
  53. Smith, P. R., Y. Gao, L. Karran, M. D. Jones, D. Snudden, and B. E. Griffin. 1993. Complex nature of the major viral polyadenylated transcripts in Epstein-Barr virus-associated tumors. J. Virol. 67:3217-3225.[Abstract/Free Full Text]
    54. 54
  54. Sugiura, M., S. Imai, M. Tokunaga, S. Koizumi, M. Uchizawa, K. Okamoto, and T. Osato. 1996. Transcriptional analysis of Epstein-Barr virus gene expression in EBV-positive gastric carcinoma: unique viral latency in the tumor cells. Br. J. Cancer 74:625-631.[Medline]
    55. 55
  55. Tao, Q., and K. D. Robertson. 2003. Stealth technology: how Epstein-Barr virus utilizes DNA methylation to cloak itself from immune detection. Clin. Immunol. 109:53-63.[CrossRef][Medline]
    56. 56
  56. Tong, X., R. Drapkin, D. Reinberg, and E. Kieff. 1995. The 62- and 80-kDa subunits of transcription factor II H mediate the interaction with Epstein-Barr virus nuclear protein 2. Proc. Natl. Acad. Sci. USA 92:3259-3263.[Abstract/Free Full Text]
    57. 57
  57. Tong, X., R. Drapkin, R. Yalamanchili, G. Mosialos, and E. Kieff. 1995. The Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE. Mol. Cell. Biol. 15:4735-4744.[Abstract]
    58. 58
  58. Tong, X., F. Wang, C. J. Thut, and E. Kieff. 1995. The Epstein-Barr virus nuclear protein 2 acidic domain can interact with TFIIB, TAF40 and RPA70 but not with TATA-binding protein. J. Virol. 69:585-588.[Abstract]
    59. 59
  59. Tsai, C.-N., C.-M. Lee, C.-K. Chien, S.-C. Kuo, and Y.-S. Chang. 1999. Additive effect of Sp1 and Sp3 in regulation of the ED-L1E promoter of the EBV LMP 1 gene in human epithelial cells. Virology 261:288-294.[CrossRef][Medline]
    60. 60
  60. Tun, T., Y. Hamaguchi, N. Matsunami, T. Furukawa, T. Honjo, and M. Kawaichi. 1994. Recognition sequence of a highly conserved DNA binding protein RBP-J kappa. Nucleic Acids Res. 22:965-971.[Abstract/Free Full Text]
    61. 61
  61. Usacheva, A., X. Tian, R. Sandoval, D. Salvi, D. Levy, and O. R. Colamonici. 2003. The WD motif-containing protein RACK-1 functions as a scaffold protein within the type I IFN receptor-signaling complex. J. Immunol. 171:2989-2994.[Abstract/Free Full Text]
    62. 62
  62. van Beek, J., A. A. Brink, M. B. Vervoort, M. J. van Zijp, C. J. Meijer, A. J. van den Brule, and J. M. Middeldorp. 2003. In vivo transcription of the Epstein-Barr virus (EBV) BamHI-A region without associated in vivo BARF0 protein expression in multiple EBV-associated disorders. J. Gen. Virol. 84:2647-2659.[Abstract/Free Full Text]
    63. 63
  63. Voss, M. D., A. Hille, S. Barth, A. Spurk, F. Hennrich, D. Holzer, N. Mueller-Lantzsch, E. Kremmer, and F. A. Grasser. 2001. Functional cooperation of Epstein-Barr virus nuclear antigen 2 and the survival motor neuron protein in transactivation of the viral LMP1 promoter. J. Virol. 75:11781-11790.[Abstract/Free Full Text]
    64. 64
  64. Wallberg, A. E., K. Pedersen, U. Lendahl, and R. G. Roeder. 2002. p300 and PCAF act cooperatively to mediate transcriptional activation from chromatin templates by notch intracellular domains in vitro. Mol. Cell. Biol. 22:7812-7819.[Abstract/Free Full Text]
    65. 65
  65. Wang, L., S. R. Grossman, and E. Kieff. 2000. Epstein-Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter. Proc. Natl. Acad. Sci. USA 97:430-435.[Abstract/Free Full Text]
    66. 66
  66. Woisetschlaeger, M., X. W. Jin, C. N. Yandava, L. A. Furmanski, J. L. Strominger, and S. H. Speck. 1991. Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection. Proc. Natl. Acad. Sci. USA 88:3942-3946.[Abstract/Free Full Text]
    67. 67
  67. Woisetschlaeger, M., C. N. Yandava, L. A. Furmanski, J. L. Strominger, and S. H. Speck. 1990. Promoter switching in Epstein-Barr virus during the initial stages of infection of B lymphocytes. Proc. Natl. Acad. Sci. USA 87:1725-1729.[Abstract/Free Full Text]
    68. 68
  68. Wu, D. Y., A. Krumm, and W. H. Schubach. 2000. Promoter-specific targeting of human SWI-SNF complex by Epstein-Barr virus nuclear protein 2. J. Virol. 74:8893-8903.[Abstract/Free Full Text]
    69. 69
  69. Wu, F. Y., S. E. Wang, H. Chen, L. Wang, S. D. Hayward, and G. S. Hayward. 2004. CCAAT/enhancer binding protein {alpha} binds to the Epstein-Barr virus (EBV) ZTA protein though oligomeric interactions and contributes to cooperative transcriptional activation of the ZTA promoter though direct binding to the ZII and ZIIIB motifs during induction of the EBV lytic cycle. J. Virol. 78:4847-4865.[Abstract/Free Full Text]
    70. 70
  70. Wu, L., J. C. Aster, S. C. Blacklow, R. Lake, S. Artavanis-Tsakonas, and J. D. Griffin. 2000. MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat. Genet. 26:484-489.[CrossRef][Medline]
    71. 71
  71. Zhang, J., H. Chen, G. Weinmaster, and S. D. Hayward. 2001. Epstein-Barr virus BamHI-A rightward transcript-encoded RPMS protein interacts with the CBF1-associated corepressor CIR to negatively regulate the activity of EBNA2 and NotchIC. J. Virol. 75:2946-2956.[Abstract/Free Full Text]
    72. 72
  72. Zhang, L., and J. S. Pagano. 2000. Interferon regulatory factor 7 is induced by Epstein-Barr virus latent membrane protein 1. J. Virol. 74:1061-1068.[Abstract/Free Full Text]
    73. 73
  73. Zhang, L., and J. S. Pagano. 1997. IRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency. Mol. Cell. Biol. 17:5748-5757.[Abstract]
    74. 74
  74. Zhou, S., M. Fujimuro, J. J. Hsieh, L. Chen, and S. D. Hayward. 2000. A role for SKIP in EBNA2 activation of CBF1-repressed promoters. J. Virol. 74:1939-1947.[Abstract/Free Full Text]
    75. 75
  75. Zhou, S., M. Fujimuro, J. J.-D. Hsieh, L. Chen, A. Miyamoto, G. Weinmaster, and S. D. Hayward. 2000. SKIP, a CBF1-associated protein, interacts with the ankyrin repeat domain of NotchIC to facilitate NotchIC function. Mol. Cell. Biol. 20:2400-2410.[Abstract/Free Full Text]
    76. 76
  76. Zhu, F. X., S. M. King, E. J. Smith, D. E. Levy, and Y. Yuan. 2002. A Kaposi's sarcoma-associated herpesviral protein inhibits virus-mediated induction of type I interferon by blocking IRF-7 phosphorylation and nuclear accumulation. Proc. Natl. Acad. Sci. USA 99:5573-5578.[Abstract/Free Full Text]

Journal of Virology, February 2005, p. 1724-1733, Vol. 79, No. 3
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.3.1724-1733.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Choy, E. Y.-W., Siu, K.-L., Kok, K.-H., Lung, R. W.-M., Tsang, C. M., To, K.-F., Kwong, D. L.-W., Tsao, S. W., Jin, D.-Y. (2008). An Epstein-Barr virus-encoded microRNA targets PUMA to promote host cell survival. JEM 205: 2551-2560 [Abstract] [Full Text]  
  • Edwards, R. H., Marquitz, A. R., Raab-Traub, N. (2008). Epstein-Barr Virus BART MicroRNAs Are Produced from a Large Intron prior to Splicing. J. Virol. 82: 9094-9106 [Abstract] [Full Text]  
  • Martin, H. J., Lee, J. M., Walls, D., Hayward, S. D. (2007). Manipulation of the Toll-Like Receptor 7 Signaling Pathway by Epstein-Barr Virus. J. Virol. 81: 9748-9758 [Abstract] [Full Text]  
  • Xing, L., Kieff, E. (2007). Epstein-Barr Virus BHRF1 Micro- and Stable RNAs during Latency III and after Induction of Replication. J. Virol. 81: 9967-9975 [Abstract] [Full Text]  
  • Grundhoff, A., Sullivan, C. S., Ganem, D. (2006). A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses. RNA 12: 733-750 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, H.
Right arrow Articles by Hayward, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.
Right arrow Articles by Hayward, S. D.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»