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
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ABSTRACT |
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Present address: Department of Biology, Brunel University, Uxbridge, UK.
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INTRODUCTION |
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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.
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METHODS |
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Plasmid construction.
Plasmid SK (containing EBV 137908138989 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 138173138989 in pCAT) was made by cutting SK with HindIII and BglII, blunting the ends with T4 DNA polymerase and religation. Plasmid CK (containing EBV 138289138989 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 137908138720 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 138926138941 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.
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RESULTS |
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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.
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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-
B site), the latter being downstream of exon 1. The B site showed protection, the NF-
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).
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DISCUSSION |
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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.
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REFERENCES |
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Brooks, L. A., Lear, A. L., Young, L. S. & Rickinson, A. B. (1993). Transcripts from the EpsteinBarr virus BamHI A fragment are detectable in all three forms of virus latency. J Virol 67, 31823190.[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, 599606.[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, 193201.[Medline]
Chen, H., Smith, P., Ambinder, R. F. & Hayward, S. D. (1999). Expression of EpsteinBarr virus BamHI-A rightward transcripts in latently infected B cells from peripheral blood. Blood 93, 30263032.
Chen, H., Lee, J. M., Zong, Y., Borowitz, M., Ng, M. H., Ambinder, R. F. & Hayward, S. D. (2001). Linkage between STAT regulation and EpsteinBarr virus gene expression in tumors. J Virol 75, 29292937.
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 EpsteinBarr virus. Int J Cancer 83, 121126.[CrossRef][Medline]
Farrell, P. J. (1989). The EpsteinBarr virus genome. Adv Viral Oncology 8, 103132.
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, 248254.[Medline]
Gilligan, K., Sato, H., Rajadurai, P., Busson, P., Young, L., Rickinson, A., Tursz, T. & Raab-Traub, N. (1990). Novel transcription from the EpsteinBarr virus terminal EcoRI fragment, DIJhet, in a nasopharyngeal carcinoma. J Virol 64, 49484956.[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 EpsteinBarr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo. J Virol 65, 62526259.[Medline]
Griffin, B. E. & Xue, S. A. (1998). EpsteinBarr virus infections and their association with human malignancies: some key questions. Ann Med 30, 249259.[Medline]
Heinemeyer, T., Wingender, E., Reuter, I. & 9 other authors (1998). Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26, 362367.
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, 26392651.[Abstract]
Karran, L., Gao, Y., Smith, P. R. & Griffin, B. E. (1992). Expression of a family of complementary-strand transcripts in EpsteinBarr virus-infected cells. Proc Natl Acad Sci U S A 89, 80588062.[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 EpsteinBarr virus: a critical role for antigen expression. J Virol 72, 66146620.
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, 27282738.[CrossRef][Medline]
Kusano, S. & Raab-Traub, N. (2001). An EpsteinBarr virus protein interacts with Notch. J Virol 75, 384395.
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, 789792.[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 EpsteinBarr virus genomes. J Virol 76, 41134118.
Raab-Traub, N., Rajadurai, P., Flynn, K. & Lanier, A. P. (1991). EpsteinBarr virus infection in carcinoma of the salivary gland. J Virol 65, 70327036.[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, 382532.[Abstract]
Robertson, K. D., Manns, A., Swinnen, L. J., Zong, J. C., Gulley, M. L. & Ambinder, R. F. (1996). CpG methylation of the major EpsteinBarr virus latency promoter in Burkitt's lymphoma and Hodgkin's disease. Blood 88, 31293136.
Sadler, R. H. & Raab-Traub, N. (1995). Structural analyses of the EpsteinBarr virus BamHI A transcripts. J Virol 69, 11321141.[Abstract]
Salamon, D., Takacs, M., Ujvari, D., Uhlig, J., Wolf, H., Minarovits, J. & Niller, H. H. (2001). ProteinDNA binding and CpG methylation at nucleotide resolution of latency-associated promoters Qp, Cp, and LMP1p of EpsteinBarr virus. J Virol 75, 25842596.
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 EpsteinBarr virus. J Virol 74, 30823092.
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 EpsteinBarr virus infection. J Virol 75, 35373546.
Sugiura, M., Imai, S., Tokunaga, M., Koizumi, S., Uchizawa, M., Okamoto, K. & Osato, T. (1996). Transcriptional analysis of EpsteinBarr virus gene expression in EBV-positive gastric carcinoma: unique viral latency in the tumour cells. Br J Cancer 74, 625631.[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, 380392.[Abstract]
Wensing, B., Stuhler, A., Jenkins, P., Hollyoake, M., Karstegl, C. E. & Farrell, P. J. (2001). Variant chromatin structure of the oriP region of EpsteinBarr virus and regulation of EBER1 expression by upstream sequences and oriP. J Virol 75, 62356241.
Zhang, C. X., Lowrey, P., Finerty, S. & Morgan, A. J. (1993). Analysis of EpsteinBarr 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, 509514.[Abstract]
Zhang, J., Chen, H., Weinmaster, G. & Hayward, S. D. (2001). EpsteinBarr 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, 29462956.
Received 20 December 2002;
accepted 21 February 2003.