Updated Epstein–Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs of EBV

Orlando de Jesus1, Paul R. Smith1,{dagger}, Lindsay C. Spender1, Claudio Elgueta Karstegl1, Hans Helmut Niller2, Dolly Huang3 and Paul J. Farrell1

1 Ludwig Institute for Cancer Research, Faculty of Medicine, Imperial College, St Mary's Campus, Norfolk Place, London W2 1PG, UK
2 Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, D-93053 Regensburg, Germany
3 Department of Clinical Oncology, Sir Y.K. Pao Centre for Cancer, The Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region

Correspondence
Paul Farrell
p.farrell{at}imperial.ac.uk


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Two sequences required for activity of the Epstein–Barr virus BART RNA promoter in transfection assays have been identified by site-directed mutagenesis. One contains a consensus AP-1 site; the other has some similarity to Ets and Stat consensus binding sites. Candidate sequences were suggested by mapping a region of unmethylated DNA in EBV around the BART promoter followed by in vivo footprinting the promoter in the C666-1 nasopharyngeal carcinoma cell line, which expresses BART RNAs. The data are presented in the context of a revised EBV DNA sequence, known as EBV wt, that is proposed as a future standard sequence for EBV.

{dagger}Present address: Department of Biology, Brunel University, Uxbridge, UK.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Epstein–Barr virus (EBV) contains about 85 genes but only a few of these are expressed in the EBV-associated human cancers that occur in immunocompetent patients. Since it has become clear that many of the established immortalizing proteins of EBV are not expressed in human tumours, attention has turned to those viral genes that are expressed in cancers for interpretation of the oncogenic properties of EBV. The BART/CST (BamHI A rightward transcript/complementary strand transcript) RNAs were originally identified in nasopharyngeal carcinoma (NPC) samples (Hitt et al., 1989), although the transcripts have also been detected at low level in some Burkitt's lymphoma and B lymphoblastoid cell lines (Brooks et al., 1993; Chen et al., 1992; Gilligan et al., 1990; Griffin & Xue, 1998; Karran et al., 1992; Raab-Traub et al., 1991; Zhang et al., 1993). There is also evidence for BART RNAs in EBV-positive gastric cancer (Sugiura et al., 1996), Hodgkin's Disease (Zhang et al., 2001) and in normal EBV persistence (Chen et al., 1999; Gilligan et al., 1991; Kienzle et al., 1998), although the BART region can be deleted from the viral genome without any notable effect on B cell immortalization by EBV. The protein products from the BART RNAs have not yet been fully characterized but several potential products of the various spliced forms of BART RNA have been analysed (Smith et al., 2000) including RPMS1, A73 and BARF0 (RK-BARF0). Biochemical activities of these proteins have been identified that could be relevant to the role of the virus in cancer (Kusano & Raab-Traub, 2001; Smith et al., 2000; Zhang et al., 2001).

The splicing of the BART RNAs is complex (Sadler & Raab-Traub, 1995), with at least 16 different, partly overlapping exons identified already in cDNA. The main full-length cDNA isolated so far (Smith et al., 2000) was able to express the RPMS1 protein when transfected and there was evidence that such RNA constitutes a significant proportion of the BART RNA expressed in the C15 NPC xenograft tumour, which has relatively high expression of the BART RNAs. The transcription start was determined and a plasmid named SK containing EBV sequences from 442 nt upstream of the transcription start, through the first exon and some of the first intron was found to express the correctly initiated and spliced first exon of the BART RNA (Smith et al., 2000).

We now identify genomic sequences around the BART RNA first exon which are protected from DNA methylation in C15 NPC tumour cells, characterize sequences required for transcription from the promoter and demonstrate in vivo footprinting of those sites in a cell line derived from NPC that maintains episomal EBV. Description of exons of the BART RNAs has been complicated by the fact that the gene spans the region of EBV deleted in the B95-8 strain that was sequenced initially (Baer et al., 1984). A revised EBV sequence called EBV wt, renumbered to include the B95-8 deletion and various other corrections, is henceforth used to describe the virus and is proposed as a new standard reference sequence for EBV. The revised EBV wt sequence numbering is used to describe all EBV features in this paper.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell lines.
293 and C666-1 cells (Cheung et al., 1999) were cultured in DMEM or RPMI with 10 % foetal calf serum. Transfection into 293 cells using the calcium phosphate method and RPA analysis were as described previously (Spender et al., 2001; Wensing et al., 2001).

Plasmid construction.
Plasmid SK (containing EBV 137908–138989 in pCAT) was made by cloning an SphI to KpnI EBV restriction fragment between the SphI and HincII sites of pBluescript (Stratagene), the KpnI site having been made blunt with T4 DNA polymerase. The EBV fragment was excised using the HindIII and XbaI sites in the Bluescript polylinker and cloned between the HindIII and XbaI sites of pCAT-Basic (Promega). Plasmid BK (containing EBV 138173–138989 in pCAT) was made by cutting SK with HindIII and BglII, blunting the ends with T4 DNA polymerase and religation. Plasmid CK (containing EBV 138289–138989 in pCAT-Basic) was made by Pfu polymerase PCR from SK and cloned between the SphI and XbaI sites of pCAT-Basic. Plasmid SS (containing EBV 137908–138720 in pCAT) was made by cloning an SphI to SspI EBV restriction fragment between the SphI and HincII sites of pBluescript, the SspI site having been made blunt with T4 DNA polymerase. The EBV fragment was excised using the HindIII and XbaI sites in the Bluescript polylinker and cloned between the HindIII and XbaI sites of pCAT-Basic. Mutations were introduced into BK and CK using the Quikchange kit (Promega). The A site was changed from TGAGTCA to TGAGGCA, the B site was changed from TACCCGGAA to TACCCGGGC and the N site was deleted. For this, EBV nucleotides 138926–138941 were deleted resulting in a sequence CAGTGTGC. All plasmids were sequence verified in the BART promoter region.

DMS interference footprinting and methylation analysis.
In vivo dimethyl sulphate (DMS) interference footprinting was performed as described previously (Niller et al., 2002). The C15 tumour was propagated in nude mice (Busson et al., 1988) and DNA was extracted by proteinase K digestion and phenol extraction. For methylation analysis, C15 tumour DNA was digested with either HpaII or MspI, electrophoresed on a 1 % agarose gel and Southern blots were hybridized with the probes indicated in the legend to Fig. 1, labelled by random priming.



View larger version (74K):
[in this window]
[in a new window]
 
Fig. 1. Methylation of C15 EBV DNA spares the pBART promoter. DNA from C15 tumour was digested with either HpaII (H) or MspI (M) and analysed by Southern blotting using probes corresponding to the indicated regions 1–8 or a probe for the LMP2 gene region (LMP2). The positions of MspI/HpaII sites interpreted according to the EBVwt sequence to be either unmethylated or methylated are indicated as either U or M respectively or U/M in case of substantial partial methylation. Sites that are very close together are indicated by a single U or M for clarity; these are bracketed together in the following list of positions of the sites in the EBVwt genome: M137349, M137570, M(137802, 137857), M(138058, 138072), U(138335, 138350), U138733, U(138848, 138875, 138894), U139013, U139148, U/M(139320, 139413), M 139714, M140071. Probes were approximately 1 (137000–137640), 2 (137640–137920), 3 (137920–138180), 4 (138180–138740), 5 (138740–139010), 6 (139010–139750), 7 (139530–140430), 8 (140430–140850); LMP2 is an LMP2A cDNA probe. The map positions of the BILF2 open reading frame, exons I, IA and IB of the BART RNAs and the EBV content of the SK plasmid are shown above against a scale in kb corresponding to the EBV wt map. The predicted MspI fragments are marked with filled arrowheads, high molecular mass DNA resistant to HpaII is marked with filled circles and the partially methylated fragments are marked with open arrowheads.

 
Gel retardation assay (EMSA).
Double stranded oligonucleotides used were as follows. A site probe, CTAAATGAGTCATTCCTAA; mutated probe, CTAAATGAGGCATTCCTAA. B site probe, GCCATACCCGGAAGAGGAG; mutated probe, GCCATACCCGGGCGAGGAG. N site probe, GTAGGGGCCTCCACCTAGGT. Oligonucleotides were end-labelled with T4 polynucleotide kinase. To prepare nuclear extracts, cells were scraped and washed in PBS then resuspended in Buffer A [10 mM HEPES, 1·5 mM MgCl2, 10 mM KCl, 0·5 mM DTT, 0·5 mM PMSF, 0·1 % NP40, 1x protease inhibitor cocktail (Boehringer Mannheim)] and left on ice for 5 min. After brief centrifugation the supernatant was removed and the nuclei resuspended in Buffer B (25 % glycerol, 20 mM HEPES, 420 mM NaCl, 1·5 mM MgCl2, 10 mM KCl, 0·2 mM EDTA, 1 mM DTT, 0·5 mM PMSF, 1x protease inhibitor cocktail) and mixed at 4 °C for 15 min. Cell debris was removed by centrifugation and protein concentration determined using the Bio-Rad DC protein assay. For each reaction, 5 µg of nuclear extract was incubated at 25 °C for 5 min with a mixture of 2·5 µl BSA (2 mg/ml), 2 µl poly(dI:dC) (2 mg/ml) (Sigma), 0·5 µl 200 mM DTT and 5 µl Buffer D (20 mM HEPES pH 7·9, 100 mM KCl, 0·2 mM EDTA, 0·5 mM PMSF, 0·5 mM DTT, 20 % glycerol, 1x protease inhibitor cocktail. When competitor oligonucleotides were used, they were added at this point. The relevant 32P-labelled double stranded oligonucleotide was then added (0·4 ng per reaction) and the mixture was incubated at 25 °C for 30 min. Samples were electrophoresed on 4 % polyacrylamide gels in 0·3x Tris/borate/EDTA; the gel was then dried and data were collected on the phosphorimager.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
New reference sequence for EBV
The prototype DNA sequence for EBV has been the B95-8 strain, accession no. V01555 (Baer et al., 1984). The revised EBV wt map and sequence file (accession no. AJ507799) used here have several advantages over the Baer et al. sequence. The B95-8 deletion sequence determined from Raji EBV has been inserted to give a more wild-type genome and the number of major internal repeat copies has been reduced from 11 to 7, which is more representative. A recently discovered single nucleotide error in the BcRF1 open reading frame (W. Amon & P. J. Farrell, unpublished) has been corrected and the annotation has been improved and brought into line with current standards. This sequence, known as EBV wt, is available with corrected and updated annotation from data libraries with accession no. AJ507799 and a corresponding genome map can also be downloaded from the http://www.med.ic.ac.uk/ludwig/ebv.htm website. The continuous sequence will allow a simpler description of the BART RNAs which cross the B95-8 deletion. The promoter for these RNAs is the topic of this paper.

EBV DNA is unmethylated around exon I of the BART gene
Several studies have shown that most of the EBV genome DNA is methylated in tumour cell lines that have a latent infection with EBV (e.g. Robertson et al., 1996; Salamon et al., 2001). Absence of DNA methylation in a region of the EBV genome can be an indicator of locations where transcription factors required for promoter activity may be bound during EBV latency. Comparison of restriction digestion by HpaII and MspI on Southern blots of C15 tumour EBV DNA indicated a region of mostly unmethylated DNA extending from about 138200 to 139200 on the EBV wt map (Fig. 1). For example, probes 4 and 5 in the hypomethylated region give mostly the same sized bands with the two enzymes whereas with probes 1 and 2 show no digestion with the methylation-sensitive HpaII enzyme. The region of hypomethylation extends from just upstream of the transcription start to a significant distance downstream of exon 1 (Fig. 1). We previously showed (Smith et al., 2000) that promoter activity could be observed in the plasmid SK (Fig. 1) in transfection assays.

C666-1 cells express BART RNAs – genomic footprinting the BART promoter
Most of our previous investigation of EBV gene expression in NPC has used the C15 xenograft because there has been a lack of NPC cell lines that retain their EBV. The recently described C666-1 line (Cheung et al., 1999) is derived from an NPC, retains its EBV and has been shown to have a restricted latent pattern of EBV gene expression. The cells make EBNA-1 protein but not EBNA-2 or LMP1 (Cheung et al., 1999). Using similar RPA probes to those applied previously in C15 (Smith et al., 2000), BART RNA expression was also readily detected in C666-1 RNA. Correctly spliced exon I and the boundary between exons VIIA and VIIB were demonstrated (Fig. 2). In each case, RNA that was not spliced at the splice junction was also detected; this might reflect partly spliced nuclear RNA (total cell RNA was used for these RPA experiments) or may indicate a heterogeneity of splicing in the BART RNAs. The unspliced signal could not be derived from viral DNA contaminating the RNA because the 200 nt band in the exon I RPA corresponds to the length of correctly initiated RNA unspliced at the exon I 3' end rather than the whole EBV content of the probe (379 nt), which would be protected by viral genomic DNA.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2. RPA analysis of C666-1 RNA for (A) BART RNA exon I. The 125 nt protected fragment corresponding to exon I is marked with an arrow. (B) The exon VIIA to VIIB splice is shown by the 340 nt protected fragment. Probes were as described previously (Smith et al., 2000); track P is input probe, Y is yeast RNA negative control. (C) Gardella gel analysis (Gardella et al., 1984) of EBV DNA in C666-1, Akata and LCL-C (a B95-8 EBV LCL) cell lines using a BamHI W probe. Track EBV contains B95-8 EBV virions as a marker for linear EBV DNA; DG75 is an EBV negative control line.

 
The C666-1 line is unusual in the sense that there has been great difficulty in obtaining an NPC cell line that retains its EBV, so we also checked that the EBV genome was in the normal episomal state and had not suffered major deletions. Southern blotting BamHI digests of C666-1 DNA (data not shown) revealed the normal BamHI fragments C, W, K and A, which are widely distributed along the genome, indicating no obvious major deletions. Gardella gel analysis (Gardella et al., 1984) showed the typical episomal EBV found also in a B95-8 lymphoblastoid cell line (LCL C) and in the Akata Burkitt's lymphoma cell line (Fig. 2). The lymphoid lines have a small fraction of cells spontaneously in the productive cycle giving some linear EBV DNA but this was only present at a very low level in C666-1, consistent with the latent cycle protein expression pattern reported previously (Cheung et al., 1999).

To identify likely binding sites for transcription factors within C666-1 EBV corresponding to the SK plasmid region, DMS interference in vivo footprinting was applied to the C666-1 cells. The interpretation of this type of data is sensitive to sequence variation relative to the prototype EBV-wt sequence so this region of C15, C666-1 and Akata EBV was first sequenced. A few sites of variation were detected, summarized in the legend to Fig. 3, but there was no variation from the EBV wt sequence in the sites identified by the footprinting. The footprinting showed several sites of either protection or enhanced cleavage relative to the equivalent naked plasmid DNA. These are shown in Fig. 3 and were named A (includes an AP1 consensus binding site TGAGTCA), B (sequence similar to an Ets or Stat consensus site) and N (some similarity to an NF-{kappa}B site), the latter being downstream of exon 1. The B site showed protection, the NF-{kappa}B site had enhanced cleavage and the A site had both protection and enhanced cleavage. The positions of these sites relative to the transcription map are shown in Fig. 4(A). The footprinting was done in C666-1 cells but subsequent transfection assays for BART promoter activity (Fig. 4) were performed in 293 cells because of their higher transfection efficiency, so extracts of both 293 and C666-1 cells were tested for binding oligonucleotides containing the A, B or N site. Clear binding of the A and B sites was observed by EMSA (Fig. 3B) with both 293 and C666-1 extract and this was specific since it was competed by an excess of the same oligonucleotide but not by an oligonucleotide in which some nucleotides had been mutated (the same mutations as used below in functional assays of the promoter). A single major A site complex was observed with C666-1 extract but several complexes were seen with 293 extract (Fig. 3B). The most specific of these (arrowed), as determined by competitor oligonucleotides, migrated close to the position of the C666-1 complex. It was already well established, e.g. Kirch et al. (1999), that 293 cells contain AP-1 activity which can bind to the same sequence that is present in site A. An oligonucleotide containing the N site was also tested with extracts from 293 and C666-1 cells but no specific binding was observed in the EMSA (data not shown).



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 3. (A) DMS interference footprints corresponding to Site A, B and N. Track Fp is the footprint and track G is the naked DNA control. The positions of the protection or enhanced cleavage are indicated by * on the local EBV sequences. Sequence differences between EBVwt and C666-1 were at 138283 (T to C), 138577 (T to G), 138580 (A to C), 138699 (G to T), 138723 (T to C), 138813 (T to G), 138870-138872 (TCC deleted) and 138987 (C to A). Akata and C15 also had these changes from EBV wt except for the TCC deletion. (B) Gel retardation assay of protein binding to the A or B site using wild-type probe (wt) or mutated site probe (mut) with extract from 293 or C666-1 cells, as indicated. The mutation was the same as in the expression assays (Fig. 4). Tracks 0 are probe alone; 1–3 have cell extract. Tracks 2 also included 100-fold excess of the unlabelled wild-type site oligonucleotide and tracks 3 had 100-fold excess of the unlabelled mutant site oligonucleotide. Specific complexes are indicated by the arrows.

 


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4. (A) The EBV content of the plasmids SK, BK, CK and SS is shown beneath a map of BART exon I. The positions of the A, B and N sites are shown. (B) The indicated plasmids (mutated at the A, B or N sites, where indicated) were transfected into 293 cells and RNA was harvested 24 h later. RPA results, as in Fig. 1, for the 125 nt exon I protected fragment and a GAPDH control are shown. (C) The data from part B and another similar experiment were quantified from the phosphorimager. The exon I values, normalized for transfection efficiency (using a cotransfected {beta}-galactosidase reporter) and for RNA amount using the GAPDH values are plotted as a percentage of the BK value. The mean values of the two experiments are shown in the histogram.

 
To determine more precisely the sequences required for BART promoter activity, deletions were made in the SK plasmid and site-directed mutations were made at the locations identified by the in vivo footprinting (Fig. 4A). Mutations were introduced into the A and B sites and the N site was deleted from the BK plasmid. The plasmids were transfected into 293 cells and resulting RNA was assayed by RPA for exon 1 of the BART RNAs (Fig. 4B). The results were normalized relative to an RPA assay for GAPDH (Fig. 4B) and the results quantified (Fig. 4C) from the phosphorimager data. The results show that truncation of the plasmid down to BK or CK gave about twice the amount of exon 1 RNA as plasmid SK. Mutation of either the A or B site within BK caused modest reductions in expression but mutation of both A and B sites substantially reduced expression (about 10-fold). Consistent with this, mutation of the B site reduced CK expression (the A site is not present in the CK plasmid). Deletion of the N site either by truncation in the SS plasmid or by localized deletion in BK caused only a small reduction in exon 1 expression.


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The RNA mapping data shown here have further confirmed that the BART exon I starting transcription at 138350 is a significant point of initiation of BART family RNAs. The previously reported 5' end was confirmed in C666-1 cells (Fig. 2A). One surprising feature of the DNA methylation study of C15 EBV DNA reported here is that the unmethylated region of DNA extends significantly downstream of exon I. This suggests that proteins may be bound to this region during latent persistence of the virus in the C15 tumour cells, preventing DNA methylation. The downstream unmethylated region could represent components of the BART promoter, other promoters so far unmapped or other genetic functions within this region of DNA. Previous RNA mapping in B95-8 cells (Farrell, 1989) recorded poorly characterized leftward RNAs that might originate from this region and it remains unclear whether the A73 type of BART RNA (Smith et al., 2000) initiates at the normal BART exon I, so there are candidate RNAs that might come from a novel promoter in this region yet to be characterized. On the other hand, there is some evidence that could be consistent with downstream promoter elements in the BART promoter. To analyse the BART promoter, we used RPA assays on the SK, BK and CK constructs because simple fusion of the upstream region (which would normally be expected to contain the promoter sequence) to a CAT reporter gave very little CAT activity (data not shown). Downstream promoter elements are one possible explanation of this. DMS interference footprinting in the C666-1 cell line showed enhanced cleavage at site N in the downstream region, indicating a distortion of the normal DNA structure at that point but no other protein binding was observed directly. These possibilities therefore remain to be resolved.

Although the cells grow relatively slowly, the C666-1 cell line seems to be a valuable system for studying EBV gene expression in epithelial cells. It appears to contain a relatively wild-type EBV, retains the EBV in culture and expresses the BART RNAs. DMS interference footprinting suggested Sites A and B upstream of exon I that might be involved in BART expression and mutation of both of these sites substantially reduced activity of the BART promoter in a transfection assay. Site A contains a perfect match to the AP-1 consensus site that has been shown previously to bind c-Jun/c-Fos and the mutation we introduced to site A is known to prevent c-Jun/c-Fos from binding (Risse et al., 1989). A common specific EMSA band was obtained with both 293 and C666-1 cells so it is likely that this contributes to activity of the promoter in both cell lines. The factor that binds to the B site is less certain. Scanning the sequence with the TFMATRIX transcription factor binding site database (Heinemeyer et al., 1998) suggests imperfect matches to NRF-2 (93 %), c-Ets (87 %) and a STAT consensus, STATx (86 %). These are widely expressed factors with several family members and overlapping binding specificities; it is difficult to be certain which factors are the functional ones on the B site but we have demonstrated that there is a single major complex detected in EMSA analysis with this site using 293 and C666-1 cells and that the mutation of the site that prevented activity of the promoter also prevented complex formation. A factor containing NRF-2 is perhaps the most likely since this is expressed in many cell types (NRF-2 has been purified from HeLa cells; Virbasius et al., 1993). NRF-2 was originally studied as part of GABP, a factor involved in herpes simplex virus immediate early gene expression (LaMarco et al., 1991), but it is involved in expression of the cytochrome c oxidase gene and the binding site in the rat cytochrome c oxidase gene is almost identical to site B in the EBV BART promoter. Antibodies to NRF-2 are not available to test this directly. It has been proposed that Stat 3 is a major regulator of EBV latent cycle promoters in epithelial cells (Chen et al., 2001) based on the Qp and LMP1 promoters but we could find no evidence for Stat 3 binding to the B site or for binding of phosphorylated Stat 1 (data not shown).

These results are the first detailed analysis of sequences required for expression of the BART RNAs. They will provide an opportunity to identify cell factors that control expression of the BART RNAs and we have also shown that C666-1 will be a valuable system in which to investigate the BART genes since it contains an apparently normal episomal EBV genome and expresses the BART RNAs.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baer, R., Bankier, A. T., Biggin, M. D. & 9 other authors (1984). DNA sequence and expression of the B95-8 Epstein–Barr virus genome. Nature 310, 207–211.[Medline]

Brooks, L. A., Lear, A. L., Young, L. S. & Rickinson, A. B. (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]

Busson, P., Ganem, G., Flores, P. & 7 other authors (1988). Establishment and characterization of three transplantable EBV-containing nasopharyngeal carcinomas. Int J Cancer 42, 599–606.[Medline]

Chen, H. L., Lung, M. M., Sham, J. S., Choy, D. T., Griffin, B. E. & Ng, M. H. (1992). Transcription of BamHI-A region of the EBV genome in NPC tissues and B cells. Virology 191, 193–201.[Medline]

Chen, H., Smith, P., Ambinder, R. F. & Hayward, S. D. (1999). Expression of Epstein–Barr virus BamHI-A rightward transcripts in latently infected B cells from peripheral blood. Blood 93, 3026–3032.[Abstract/Free Full Text]

Chen, H., Lee, J. M., Zong, Y., Borowitz, M., Ng, M. H., Ambinder, R. F. & Hayward, S. D. (2001). Linkage between STAT regulation and Epstein–Barr virus gene expression in tumors. J Virol 75, 2929–2937.[Abstract/Free Full Text]

Cheung, S. T., Huang, D. P., Hui, A. B., Lo, K. W., Ko, C. W., Tsang, Y. S., Wong, N., Whitney, B. M. & Lee, J. C. (1999). Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein–Barr virus. Int J Cancer 83, 121–126.[CrossRef][Medline]

Farrell, P. J. (1989). The Epstein–Barr virus genome. Adv Viral Oncology 8, 103–132.

Gardella, T., Medveczky, P., Sairenji, T. & Mulder, C. (1984). Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis. J Virol 50, 248–254.[Medline]

Gilligan, K., Sato, H., Rajadurai, P., Busson, P., Young, L., Rickinson, A., Tursz, T. & Raab-Traub, N. (1990). Novel transcription from the Epstein–Barr virus terminal EcoRI fragment, DIJhet, in a nasopharyngeal carcinoma. J Virol 64, 4948–4956.[Medline]

Gilligan, K. J., Rajadurai, P., Lin, J. C., Busson, P., Abdel-Hamid, M., Prasad, U., Tursz, T. & Raab-Traub, N. (1991). Expression of the Epstein–Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo. J Virol 65, 6252–6259.[Medline]

Griffin, B. E. & Xue, S. A. (1998). Epstein–Barr virus infections and their association with human malignancies: some key questions. Ann Med 30, 249–259.[Medline]

Heinemeyer, T., Wingender, E., Reuter, I. & 9 other authors (1998). Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26, 362–367.[Abstract/Free Full Text]

Hitt, M. M., Allday, M. J., Hara, T., Karran, L., Jones, M. D., Busson, P., Tursz, T., Ernberg, I. & Griffin, B. E. (1989). EBV gene expression in an NPC-related tumour. EMBO J 8, 2639–2651.[Abstract]

Karran, L., Gao, Y., Smith, P. R. & Griffin, B. E. (1992). Expression of a family of complementary-strand transcripts in Epstein–Barr virus-infected cells. Proc Natl Acad Sci U S A 89, 8058–8062.[Abstract]

Kienzle, N., Sculley, T. B., Poulsen, L., Buck, M., Cross, S., Raab-Traub, N. & Khanna, R. (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]

Kirch, H. C., Flaswinkel, S., Rumpf, H., Brockmann, D. & Esche, H. (1999). Expression of human p53 requires synergistic activation of transcription from the p53 promoter by AP-1, NF-kappaB and Myc/Max. Oncogene 18, 2728–2738.[CrossRef][Medline]

Kusano, S. & Raab-Traub, N. (2001). An Epstein–Barr virus protein interacts with Notch. J Virol 75, 384–395.[Abstract/Free Full Text]

LaMarco, K., Thompson, C. C., Byers, B. P., Walton, E. M. & McKnight, S. L. (1991). Identification of Ets- and notch-related subunits in GA binding protein. Science 253, 789–792.[Medline]

Niller, H. H., Salamon, D., Uhlig, J., Ranf, S., Granz, M., Schwarzmann, F., Wolf, H. & Minarovits, J. (2002). Nucleoprotein structure of immediate-early promoters Zp and Rp and of oriLyt of latent Epstein–Barr virus genomes. J Virol 76, 4113–4118.[Abstract/Free Full Text]

Raab-Traub, N., Rajadurai, P., Flynn, K. & Lanier, A. P. (1991). Epstein–Barr virus infection in carcinoma of the salivary gland. J Virol 65, 7032–7036.[Medline]

Risse, G., Jooss, K., Neuberg, M., Bruller, H. J. & Muller, R. (1989). Asymmetrical recognition of the palindromic AP1 binding site (TRE) by Fos protein complexes. EMBO J 8, 3825–32.[Abstract]

Robertson, K. D., Manns, A., Swinnen, L. J., Zong, J. C., Gulley, M. L. & Ambinder, R. F. (1996). CpG methylation of the major Epstein–Barr virus latency promoter in Burkitt's lymphoma and Hodgkin's disease. Blood 88, 3129–3136.[Abstract/Free Full Text]

Sadler, R. H. & Raab-Traub, N. (1995). Structural analyses of the Epstein–Barr virus BamHI A transcripts. J Virol 69, 1132–1141.[Abstract]

Salamon, D., Takacs, M., Ujvari, D., Uhlig, J., Wolf, H., Minarovits, J. & Niller, H. H. (2001). Protein–DNA binding and CpG methylation at nucleotide resolution of latency-associated promoters Qp, Cp, and LMP1p of Epstein–Barr virus. J Virol 75, 2584–2596.[Abstract/Free Full Text]

Smith, P., de Jesus, O., Turner, D., Hollyoake, M., Elgueta Karstegl, C., Griffin, B., Karran, L., Wang, Y., Hayward, S. & Farrell, P. (2000). Structure and coding content of CST (BART) family RNAs of Epstein–Barr virus. J Virol 74, 3082–3092.[Abstract/Free Full Text]

Spender, L. C., Cornish, G. H., Rowland, B., Kempkes, B. & Farrell, P. J. (2001). Direct and indirect regulation of cytokine and cell cycle proteins by EBNA-2 during Epstein–Barr virus infection. J Virol 75, 3537–3546.[Abstract/Free Full Text]

Sugiura, M., Imai, S., Tokunaga, M., Koizumi, S., Uchizawa, M., Okamoto, K. & Osato, T. (1996). Transcriptional analysis of Epstein–Barr virus gene expression in EBV-positive gastric carcinoma: unique viral latency in the tumour cells. Br J Cancer 74, 625–631.[Medline]

Virbasius, J. V., Virbasius, C. A. & Scarpulla, R. C. (1993). Identity of GABP with NRF-2, a multisubunit activator of cytochrome oxidase expression, reveals a cellular role for an ETS domain activator of viral promoters. Genes Dev 7, 380–392.[Abstract]

Wensing, B., Stuhler, A., Jenkins, P., Hollyoake, M., Karstegl, C. E. & Farrell, P. J. (2001). Variant chromatin structure of the oriP region of Epstein–Barr virus and regulation of EBER1 expression by upstream sequences and oriP. J Virol 75, 6235–6241.[Abstract/Free Full Text]

Zhang, C. X., Lowrey, P., Finerty, S. & Morgan, A. J. (1993). Analysis of Epstein–Barr virus gene transcription in lymphoma induced by the virus in the cottontop tamarin by construction of a cDNA library with RNA extracted from a tumour biopsy. J Gen Virol 74, 509–514.[Abstract]

Zhang, J., Chen, H., Weinmaster, G. & Hayward, S. D. (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]

Received 20 December 2002; accepted 21 February 2003.