Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, S-413 45 Gothenburg, Sweden1
Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden2
Cellular and Molecular Tumor Pathology, Cancer Centrum Karolinska, CCK R8:04, Karolinska Hospital, S-171 76 Stockholm, Sweden3
Author for correspondence: Lars Rymo. Fax +46 31 828458. e-mail lars.rymo{at}clinchem.gu.se
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Abstract |
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Introduction |
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EBNA1-specific initiation of transcription in the restricted pattern of EBV gene expression in latency I B cells and NPC cells was for some time thought to occur at Fp, the promoter in the BamHI F fragment of the EBV genome (Nonkwelo et al., 1995 ; Sample et al., 1991
; Schaefer et al., 1991
), but more recent data strongly suggest that the dominant EBNA1 gene promoter is Qp, located about 200 bp downstream of Fp in the BamHI Q region (Schaefer et al., 1995b
; Tsai et al., 1995
; Zetterberg et al., 1999
). Fp is now considered an early lytic promoter (Schaefer et al., 1995a
). A small proportion (12%) of Fp-initiated lytic transcripts is spliced to the EBNA1-encoding K exon (Zetterberg et al., 1999
). However, the majority of Fp-initiated transcripts are not spliced to this exon, at least not at the regular EBNA1 splice acceptor in BamHI K (Schaefer et al., 1995a
; Zetterberg et al., 1999
). The gene product of these transcripts has not been identified.
The regulation of Fp has been the subject of several investigations during the period when Fp was considered a latent EBNA1-specific promoter. Fp was reported to be repressed by binding of EBNA1 to sequences in BamHI Q and to be highly dependent on BamHI Q sequences resembling binding sites for the cellular transcription factors E2F-1 and leader binding protein-1 (Nonkwelo et al., 1995 ; Sung et al., 1994
). However, the measured promoter activity in these experiments most likely arose from Qp. Using a reporter construct that only contained a 146 bp fragment of Fp without sequences from BamHI Q, Bulfone-Paus et al. (1995)
identified three positive promoter-proximal cis-acting elements critical for Fp regulation: one Sp1 site and two tandem LR1 sites (Fig. 1
). In earlier work, Lear et al. (1992)
showed that Fp was upregulated following the switch from latent into lytic cycle, and raised the possibility that ZEBRA, by an unknown mechanism, could upregulate Fp. In the present investigation we corroborate that Fp is a lytic promoter and show that ZEBRA activates transcription from Fp by binding to a promoter-proximal AP-1-like site.
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Methods |
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Cell lines and induction of virus lytic cycle.
The cell lines used in this study and their phenotypic characteristics are listed in Table 1. All cell lines were propagated in RPMI 1640 (Life Technologies) supplemented with 10% foetal bovine serum (Life Technologies), penicillin and streptomycin. Induction of the virus lytic cycle in Rael and Mutu gr I was performed by the addition of 5-azacytidine (Sigma) to a concentration of 5 µM (Masucci et al., 1989
). Induction of the virus lytic cycle in Rael was also performed by transfection of 5x106 cells with 10 µg of ZEBRA expression plasmid by electroporation, as described below. Induction of the virus lytic cycle in Akata was performed by the addition of anti-IgG antibodies (DAKO) to a concentration of 0·2% (Takada & Ono, 1989
). Induction of the lytic cycle was confirmed by direct immunofluorescence using a monoclonal anti-ZEBRA antibody (M7005; DAKO).
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RNA analysis.
Cytoplasmic RNA was prepared by a standard procedure (Sambrook et al., 1989 ). Transcription initiated at Fp in the endogenous EBV genome was determined by S1 mapping using a single-stranded oligonucleotide corresponding to nucleotides 6222162253 of the L strand of B95-8 EBV DNA: 5' GATCCCCCTCCTCTCTATCCACCGCCGCCCCGG 3'. A non-homologous tail of four nucleotides was added at the 3' end of the oligonucleotide to distinguish incomplete digestion of the probe by transcripts initiated further upstream. 32P-end-labelled probe (150 fmol) was annealed for 1216 h at 37 °C with 100 µg of cytoplasmic RNA in 40 mM PIPES, pH 6·4, 1 mM EDTA, 0·4 mM NaCl and 50% formamide in a total volume of 20 µl. The hybridization mixture was treated with 750 U/ml of S1 nuclease for 45 min at 37 °C. Protected fragments were fractionated by electrophoresis in a denaturing 12% polyacrylamide gel. The expected size of a fragment protected by RNA initiated at the Fp transcription start site 62239 is 15 nucleotides.
Electrophoretic mobility shift assay (EMSA).
For preparation of ZEBRA-containing nuclear extracts, 40 aliquots, each consisting of 107 DG75 cells, were transfected with 10 µg of ZEBRA expression plasmid by electroporation as described above. Nuclear extracts were prepared 3 days after transfection, essentially as described by Dignam et al. (1983) , except that antipain (5 µg/ml), leupeptin (5 µg/ml) and aprotinin (2 µg/ml) were added to the buffer in the final homogenization and dialysis steps and PMSF was replaced with Pefabloc (0·5 mM). ZEBRA expression was verified by SDSPAGE and immunoblotting using a monoclonal anti-ZEBRA antibody (M7005; DAKO). Aliquots were frozen in liquid nitrogen and stored at -80 °C. A double-stranded, blunt-ended, synthetic oligonucleotide containing the sequence between positions 62179 and 62202 in the EBV genome, (-60/-37)Fp, was used as the probe in the EMSAs. In competition experiments, the following double-stranded consensus oligonucleotides were used: (i) AP-1 consensus: 5' CGCTTGATGACTCAGCCGGAA 3'; and (ii) sequence identical to the probe, except that the AP-1-like site was transversely mutated (AP-1 mut): 5' GGCTAGCCCAGTCGGGGGTGAGGC 3' (mutated sequence underlined). Non-specific competitor was an unrelated DNA sequence. One strand of the oligonucleotide probe was labelled with [
-32P]ATP (6000 Ci/mmol, NEN Life Science Products) using polynucleotide kinase (Boehringer Mannheim) and annealed to the complementary strand. The labelled probe was purified by electrophoresis in an 8% polyacrylamide gel in TBE (0·1 M Tris, 0·1 M boric acid, 2 mM EDTA, pH 8·3). The wet gel was autoradiographed and the DNA fragment was excised, electroeluted by isotachophoresis (Öfverstedt et al., 1984
) and precipitated. Binding reactions were carried out in a volume of 30 µl containing 10 mM TrisHCl, pH 7·5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 1 µg poly(dIdC), 5 fmol labelled probe (approximately 70000 c.p.m.) and 5 µg of crude nuclear extracts from ZEBRA-transfected DG75. In competition experiments, 3 pmol of unlabelled oligonucleotides were added into the reaction mixture. After incubation at room temperature for 30 min, the samples were loaded on a 6% polyacrylamide gel (acrylamide:bisacrylamide 29:1) in TGE (25 mM TrisHCl, 0·19 M glycine, 1 mM EDTA, pH 8·3). After electrophoresis gels were dried and autoradiographed. The supershift experiments were performed as described above for the EMSAs except that 210 µl of the respective antibody was added after the incubation at room temperature. A second incubation was carried out at 4 °C for 2 h before the samples were loaded on the gel. Antibodies used for supershift experiments were anti-ZEBRA (M7005; DAKO), anti-Sp1 (sc-59X; Santa Cruz Biotechnology Inc.) and anti-c-Jun (sc-822X; Santa Cruz Biotechnology Inc.).
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Results |
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Fp activity in endogenous virus genomes
To answer the question of whether Fp activity could be detected in the endogenous virus genome and upregulated on induction of the virus lytic cycle, S1 protection analysis was performed. An end-labelled oligonucleotide containing the Fp transcription initiation site at position 62239 was used as the probe (Fig. 2). Fp-initiated transcripts were only detected in the B95-8 cell line, which contains a significant proportion of spontaneously lytic subpopulations, and in cell lines in which the virus lytic cycle had been induced (Rael ZEBRA, Rael 5azaC, Mutu gr I 5azaC and Akata Ig), corroborating the identity of Fp as an exclusively lytic promoter.
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Discussion |
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Previous studies of regulatory elements in Fp should be carefully interpreted, since they were undertaken during a period when Fp was considered an EBNA1-specific promoter in latency I and II cells. More recent data have demonstrated that EBNA1-specific initiation of transcription in restricted latency stages arises from the downstream Q promoter (Schaefer et al., 1995b ; Tsai et al., 1995
; Zetterberg et al., 1999
). Fp was reported to be regulated by sequences in BamHI Q (Nonkwelo et al., 1995
; Sung et al., 1994
). However, the measured promoter activity in these studies most likely arose from Qp. Bulfone-Paus et al. (1995)
investigated a short Fp fragment (146 bp, resembling our p
F2CAT construct) that spanned the Fp TATA box without BamHI Q sequences. In concordance with our data, their results indicated a remarkably high activity from this short promoter fragment in EBV-positive and -negative B lymphoid cells. They identified three positive cis-acting elements: one Sp1 site and two tandem LR1 sites (Fig. 1
). The activity of the short Fp fragments in our experiments (p
F1CAT and p
F2CAT) was high in latency I B cells and EBV-negative cells and below detection level in latency III B cells, suggesting that the promoter fragments may be regulated by viral or cellular transcription factors expressed in a cell-phenotype-dependent manner. Since there are no such differences between latency I and III B cells with regard to Sp1 and LR1, we suggest that viral proteins or other cellular factors may contribute to the differential regulation. For example, latency I cells may contain additional activating factors not identified in this study, or express higher levels of activating factors, whereas latency III cells may contain a repressor(s). The significance of the differential regulation is, however, unclear since the full-length Fp, which presumably is biologically more relevant, behaves in the same manner irrespective of the original latency type, i.e. is repressed in latent cells and upregulated on induction of the virus lytic cycle. Interestingly, there was a significant activity of the short Fp fragments in the epithelial HeLa cell line, which should not contain the B cell-specific transcription factor LR1 (Bulfone-Paus et al., 1995
). Since Sp1 usually functions together with other transcription factors (Ryu et al., 1999
), it will most likely collaborate with other factors in this cell type (Bulfone-Paus et al., 1995
). The promoter-proximal AP-1-like site in the -52/-46 Fp region overlaps one of the LR1 sites (Fig. 1
). However, Bulfone-Paus et al. (1995)
ruled out a functional role for AP-1 by binding experiments and by comparing reporter gene expression in cells cultured with and without phorbol 12-myristate 13-acetate. Taken together, it seems that Sp1 and LR1, presumably together with additional factors, activate the short Fp fragments in latency I cells and EBV-negative cells and that this activity is downregulated by a repressor element(s) upstream of -136. It should be noted that two other research groups have employed p
F2CAT-like reporter plasmids for studying Fp activity and measured a low promoter activity (Schaefer et al., 1995a
; Sung et al., 1994
). In both studies the Fp transcription start site was assigned to nucleotide 62229 in the EBV-genome, 10 bp upstream of the transcription start site defined by Bulfone-Paus et al. (1995)
. In our study we detected multiple initiations between positions 62232 and 62239. The consequence of this is that the p
F2CAT-like construct used by Sung et al. (1994) did not include the major Fp transcription start sites, explaining the low promoter activity in their experiments. The construct used by Schaefer et al. (1995a)
was identical to p
F2CAT, except that it contained the luciferase reporter gene instead of CAT. Thus, there is no obvious explanation for our differing results regarding this specific question. Nevertheless, the main finding of the transient transfection experiments performed in our study was that reporter plasmids carrying larger fragments of Fp (p
F3CAT, p
F4CAT and p
F5CAT) were inactive in all examined cell lines, corroborating that Fp is silent in cells that are not induced to undergo the virus lytic cycle.
Since Fp was silent in strictly latent cells, both in endogenous genomes and in reporter plasmids carrying large fragments of the Fp regulatory region, we asked if Fp activity could be upregulated by induction of the virus lytic cycle. We have shown that endogenous Fp activity was induced by treatment of EBV-infected cells with known activators of lytic virus replication, confirming previous investigations (Lear et al., 1992 ; Schaefer et al., 1995a
; Zetterberg et al., 1999
). Moreover, the silent p
F5CAT was activated in DG75 cells in the absence of other virus proteins when co-transfected with a ZEBRA expression plasmid. The induction depended on the promoter-proximal AP-1-like site, since p
F5mutCAT was only expressed at background levels in spite of the presence of ZEBRA. Notably, the AP-1 mutation introduced in the p
F5mutCAT plasmid changed three bp in the promoter-proximal LR1 site (11 bp). However, the substitutions did not significantly reduce the basal activity of Fp in DG75 cells when introduced in p
F2CAT (data not shown). This finding also supports the conclusion drawn by Bulfone-Paus et al. (1995)
that AP-1 is not involved in the regulation of Fp. Moreover, the substitutions did not alter the nucleotides essential for LR1 binding (Dempsey et al., 1998
). EMSA and supershift experiments identified ZEBRA as a component of one of the proteinDNA complexes detected with an oligonucleotide that spanned the -60/-37 Fp region and nuclear extracts from ZEBRA-transfected DG75 cells. We identified one additional band representing protein binding to the AP-1-like site. The intensity of the band was reproducibly slightly reduced by incubation with antibodies against ZEBRA and Sp1. There is no Sp1 binding site in the -60/-37 Fp region. We could not detect the band with nuclear extracts from untransfected DG75 (data not shown). Either the reduced intensity is an unspecific phenomenon, or it might be a result of an interaction between ZEBRA and Sp1. Such an interaction has not yet been described in the literature. In conclusion, we have identified one additional ZEBRA-responsive element in the EBV genome. There are three more AP-1-like sites in the upstream regulatory region of Fp. Conceivably, these are also involved in ZEBRA-induced transactivation of Fp and initiation of the EBV lytic cascade.
A major question remains: what is the 3' end exon of the Fp-initiated transcripts? In a previous investigation of relative levels of EBNA1 gene transcripts in latent and lytic stages of infection, we demonstrated that only 12% of Fp-initiated transcripts in cells induced to virus lytic cycle splice from the U exon to the EBNA1-encoding K exon (FpQ/U/K splicing pattern) (Zetterberg et al., 1999 ). The large majority of the Fp-initiated transcripts displayed an FpQ (5275% of Fp-initiated transcripts) or FpQ/U (2447%) splicing pattern. Furthermore, we identified a BamHI K-containing lytic cycle-specific transcript, which extends upstream of the BamHI f/K cleavage site. It is possible that there is a relationship between the Fp-initiated transcripts with presently unidentified 3' end exon(s) and the lytic cycle-specific BamHI K-containing mRNA, which may give rise to EBNA1. In a study of the transregulatory effects of ZEBRA on different classes of EBV promoters, Kenney et al. (1989)
showed that ZEBRA downregulates Cp and Wp. In spite of the downregulation of latent promoters, EBNA1 is not repressed on induction of the virus lytic cycle (Rowe et al., 1992
; Weigel et al., 1985
). These data suggest that EBNA1 gene transcription in the lytic cycle may be driven by a lytic cycle-specific transcription unit, possibly initiated at Fp, which could compensate for the repression of the latent EBNA1 gene promoters. EBNA1 is known to bind to oriP sequences and enhance nuclear import of oriP-containing plasmids in transient transfection experiments, most probably through its strong nuclear localization signal (Längle-Rouault et al., 1998
). It is tempting to speculate that EBNA1 may serve an important function in the virus lytic cycle through binding to oriP in the genomes of newly packaged virions and facilitate the nuclear import of virus genomes on infection of resting B cells.
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Acknowledgments |
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References |
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Received 29 January 2002;
accepted 25 March 2002.