Regulation of Expression of the Epstein-Barr Virus
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病菌学杂志 2005年第3期
Department of Microbiology, The University of Hong Kong, Hong Kong
Viral Oncology Program, Sidney Kimmel Cancer Center
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland
Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
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.
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.
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, C/EBP?, and C/EBP 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).
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/EBP? (HC108B) or Flag-IRF7, harvested 40 h after transfection, and lysed by using a chromatin immunoprecipitation (ChIP) kit (Upstate). Anti-Flag or anti-C/EBP? 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.
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.
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.
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.
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/EBP? or C/EBP strongly activated P2-driven expression in both HeLa (Fig. 7A) and DG75 (Fig. 7B) cells, and activation by C/EBP was also shown in DG75 cells (Fig. 7B). The response of the P2 promoter to C/EBP? was comparable to that seen with the promoter for IL-6, which is known to be regulated by C/EBP? (Fig. 7C).
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, C/EBP?, and C/EBP 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. In the EMSA (Fig. 8C), a strong band and a second weak band were seen upon addition of Flag-C/EBP. 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.
Regulation of P2 by C/EBP? 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/EBP?, a Western blot was performed to examine C/EBP? protein expression in a variety of B-cell lines and epithelial cell lines (Fig. 9). Three forms of C/EBP? 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/EBP? 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/EBP? 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/EBP?, although Akata cells did express the negative LIP form of C/EBP?. Expression of C/EBP? in the in vitro-immortalized B-cell lines IB4 and LCL(III) was below the level of detection. Thus, C/EBP? 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.
Additional evidence for regulation of BARTs by C/EBP? 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/EBP? found association of C/EBP? with the P2 promoter region when the immunoprecipitations were performed with either anti-C/EBP? 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/EBP? (Fig. 10B) Expression of the endogenous C/EBP?-responsive cellular IL-6 gene was also measured and showed a sixfold increase in the C/EBP?-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/EBP? is underestimated in this assay.
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, C/EBP?, and C/EBP. 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 has been described as a regulator of the latency Cp (40). C/EBP expression is induced by treatment of Akata cells with anti-IgG antibody, and C/EBP 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 would potentially result in expression of the BARTs during lytic induction.
The endogenous P2 promoter in HeLa-Bx1 cells was upregulated by transfected C/EBP? to a similar extent as a known C/EBP? responsive gene, cellular IL-6, and association of C/EBP? with the endogenous BART promoter was also shown by ChIP analysis. The positive response of the P2 promoter to C/EBP? may contribute to the higher levels of expression of the BARTs reported in epithelial cells than in B cells. C/EBP? is an acute-phase response gene that is not constitutively expressed in the B-cell compartment (44). Western blot examination of C/EBP? 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/EBP? 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/EBP? 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.
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.
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Viral Oncology Program, Sidney Kimmel Cancer Center
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland
Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, Louisiana
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.
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.
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, C/EBP?, and C/EBP 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).
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/EBP? (HC108B) or Flag-IRF7, harvested 40 h after transfection, and lysed by using a chromatin immunoprecipitation (ChIP) kit (Upstate). Anti-Flag or anti-C/EBP? 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.
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.
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.
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.
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/EBP? or C/EBP strongly activated P2-driven expression in both HeLa (Fig. 7A) and DG75 (Fig. 7B) cells, and activation by C/EBP was also shown in DG75 cells (Fig. 7B). The response of the P2 promoter to C/EBP? was comparable to that seen with the promoter for IL-6, which is known to be regulated by C/EBP? (Fig. 7C).
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, C/EBP?, and C/EBP 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. In the EMSA (Fig. 8C), a strong band and a second weak band were seen upon addition of Flag-C/EBP. 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.
Regulation of P2 by C/EBP? 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/EBP?, a Western blot was performed to examine C/EBP? protein expression in a variety of B-cell lines and epithelial cell lines (Fig. 9). Three forms of C/EBP? 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/EBP? 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/EBP? 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/EBP?, although Akata cells did express the negative LIP form of C/EBP?. Expression of C/EBP? in the in vitro-immortalized B-cell lines IB4 and LCL(III) was below the level of detection. Thus, C/EBP? 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.
Additional evidence for regulation of BARTs by C/EBP? 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/EBP? found association of C/EBP? with the P2 promoter region when the immunoprecipitations were performed with either anti-C/EBP? 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/EBP? (Fig. 10B) Expression of the endogenous C/EBP?-responsive cellular IL-6 gene was also measured and showed a sixfold increase in the C/EBP?-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/EBP? is underestimated in this assay.
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, C/EBP?, and C/EBP. 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 has been described as a regulator of the latency Cp (40). C/EBP expression is induced by treatment of Akata cells with anti-IgG antibody, and C/EBP 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 would potentially result in expression of the BARTs during lytic induction.
The endogenous P2 promoter in HeLa-Bx1 cells was upregulated by transfected C/EBP? to a similar extent as a known C/EBP? responsive gene, cellular IL-6, and association of C/EBP? with the endogenous BART promoter was also shown by ChIP analysis. The positive response of the P2 promoter to C/EBP? may contribute to the higher levels of expression of the BARTs reported in epithelial cells than in B cells. C/EBP? is an acute-phase response gene that is not constitutively expressed in the B-cell compartment (44). Western blot examination of C/EBP? 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/EBP? 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/EBP? 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.
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.
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