Journal of General Virology (2005), 86, 1261–1267
DOI 10.1099/vir.0.80620-0
Functional interaction of Oct transcription factors with the family of repeats in Epstein–Barr virus oriP J. Almqvist,1 J. Zou,1 Y. Linderson,1 C. Borestrom,2 E. Altiok,3 H. Zetterberg,2 L. Rymo,2 S. Pettersson1 and I. Ernberg1 1
Microbiology and Tumorbiology Center (MTC), Karolinska Institute, Nobels va¨g 16, Box 280, S-171 77 Stockholm, Sweden
Correspondence I. Ernberg
2
[email protected]
Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden
3
Acibadem Genetic Diagnostic Center, Libadiye Cad, Bogazici Sitesi, Goztepe, 34724 Istanbul, Turkey
Received 18 September 2004 Accepted 26 January 2005
The family of repeats (FR) is a major upstream enhancer of the Epstein–Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.
INTRODUCTION The 172 kb Epstein–Barr virus (EBV) genome is maintained in latently infected B cells as a circular episome and replication of this episome is activated from oriP once every cell cycle in a process that involves direct binding of the EBV nuclear protein 1 (EBNA1) to oriP. EBNA1 is essential for maintenance of the EBV genome in latently infected cells and is also expressed during latency in B cells (Chen et al., 1995). EBNA1 also regulates viral transcription in latency via direct interaction with three key promoters: the latent membrane protein 1 (LMP1), C and Q promoters (LMP1p, Cp and Qp, respectively; Gahn & Sugden, 1995; Sugden & Warren, 1989; Sung et al., 1994). EBNA1 binds as a dimer to the palindromic core consensus 16 bp sequence G(A/T)TAGCATATGCTA(C/T)C, which can be found in several copies at three different sites in the EBV genome: in the dyad-symmetry element and the family of repeats (FR) in oriP upstream of Cp and downstream of Qp (Ambinder et al., 1990; Reisman & Sugden, 1986; Wysokenski & Yates, 1989; Yates et al., 1984). Due to sequence variation in the binding motif, EBNA1 has the strongest affinity for the FR and the lowest for the binding sites in Qp (Ambinder et al., 1990; Rawlins et al., 1985). In the prototype B95-8 virus, the FR comprises 20 copies of a 30 bp repeat element containing the EBNA1 core-binding site and functions as an EBNA1-dependent enhancer for Cp (La¨ngle-Rouault et al., 1998; Nilsson et al., 1993; Sugden & Warren, 1989). EBNA1 is expressed from 0008-0620 G 2005 SGM
Printed in Great Britain
Cp and is also essential for transactivation of Cp (Puglielli et al., 1996; Wysokenski & Yates, 1989). Full FR–EBNA1mediated transactivation of Cp requires at least eight EBNA1-binding sites within the FR (Zetterberg et al., 2004). Several other cis-acting transcription-regulatory elements have been identified in the regions upstream and downstream of the promoter, e.g. a glucocorticoid-responsive element (Kupfer & Summers, 1990), an EBNA2-responsive enhancer (Jin & Speck, 1992; Sung et al., 1991) and binding sites for NF-Y, Sp1, Egr-1 and members of the C/EBP transcription-factor family (Borestro¨m et al., 2003; Nilsson et al., 2001). Here, we demonstrated that members of the octamerbinding factor (Oct) family of transcription factors could bind to and activate transcription via the FR, which is of interest in relation to viral promoter regulation in B cells and epithelial cells.
METHODS Plasmids and PCR. The expression vectors Rc/CMV-EBNA1
(Levitskaya et al., 1995), pEV3S1.Oct-1 (from W. Schaffner, Institute for Molecular Biology, University of Zurich, Switzerland), WS2 (Oct-2.6) (from T. Wirth, Department of Physiological Chemistry, University of Ulm, Germany) and pEV-OBF1 (Bob.1) (from P. Matthias, Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Basel, Switzerland) are all regulated through cytomegalovirus (CMV) promoters. 1261
J. Almqvist and others The luciferase reporter vector containing the FR upstream of a thymidine kinase promoter (pT81luc-FR) was constructed by PCR amplification of FR using B95-8 DNA as template (nt 7401–8044; see Fig. 3b). This fragment was inserted in the pT81luc plasmid (Nordeen, 1988) by using the pGEM-T Easy Vector system (Promega) according to the manufacturer’s manual. The luciferase reporter vector p(oriPI/ 2170Cp)Luc was constructed by using the oriPI (FR) and 2170Cp fragments (nt 7315–8190 and 11166–11412, respectively) from the previously described pg(oriPI/2170Cp)CAT (Nilsson et al., 2001) (Fig. 1b). The fragments were inserted into the pGL3Basic vector (Promega). All constructs were verified by using an ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems). Cell lines, nuclear extracts and Western blotting. DG75 is an
EBV-negative Burkitt’s lymphoma cell line (Ben-Bassat et al., 1977). Rael is an EBV-positive Burkitt’s lymphoma cell line with a latency I expression pattern (Klein et al., 1972). The CBMI-Ral-STO cell line was obtained by in vitro infection of cord-blood cells with virus rescued from Rael cells and had a latency III expression pattern (Ernberg et al., 1989). All lymphoid cell lines were maintained as suspension cultures in RPMI 1640 medium (Sigma) supplemented with 10 % fetal bovine serum (FBS), streptomycin and penicillin (Sigma). QBI-HEK 293A cells (293A), a human embryonic kidney monolayer epithelial-cell line, were cultured in minimal essential medium (Gibco-BRL, Life Technologies) supplemented with 8 % FBS, penicillin and streptomycin.
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Nuclear extracts were prepared for electrophoretic mobility-shift assays (EMSAs) as follows. Cells were suspended in a hypotonic buffer (10 mM HEPES, 10 mM KCl, 0?1 mM EDTA, 0?1 mM EGTA). After 15 min on ice, NP-40 was added to a concentration of 0?25 %, samples were centrifuged and nuclear proteins were extracted from the pellets by using 100 ml 20 mM HEPES, 25 % glycerol, 0?4 M NaCl and 1 mM EDTA at 4 uC for 15 min. Supernatants were collected and kept at 280 uC. The protein concentration of the nuclear extracts was determined by using the Dc protein assay (Bio-Rad). Equal amounts of nuclear extract were loaded in each lane. Proteins were fractionated by SDS-PAGE (9 % gel) and transferred to nitrocellulose membranes. After blocking for 1 h at room temperature with 5 % dried milk made up in PBS/0?1 % Tween 20, the membranes were probed overnight at 4 uC with the following antibodies: anti-b-actin (diluted 1 : 5000; Sigma); anti-Oct1 (Upstates), anti-Oct-2 and anti-Bob.1 (Santa Cruz Biotechnologies) (all diluted 1 : 2000) and, for EBNA1, serum from an EBV-positive donor (diluted 1 : 4000). The secondary antibody used was conjugated to horseradish peroxidase, and bound immunocomplexes were detected by enhanced chemiluminescence (Amersham Biosciences). EMSA. EMSA was performed as follows. An oligonucleotide FR probe was end-labelled with [c-32P]dCTP and purified by native PAGE (10 % gel). Nuclear extracts were prepared as described above and 3 mg was used in each band-shift assay, in the presence of 1 mg poly(dI-dC), 1 mM Tris/HCl (pH 7?5), 100 mM NaCl, 5 mM MgCl2, 0?5 mM dithiothreitol and 2000 c.p.m. probe and mixed at room temperature. Samples were loaded on to a native polyacrylamide gel (4 %) and electrophoresed at 250 V for 1 h. After drying the gel, autoradiography was performed overnight. Unlabelled oligonucleotides were used for competition at 506 molar excess. AntiOct-1 (Upstates), anti-Oct-2, anti-Bob.1 (Santa Cruz Biotechnology) and anti-Otx-1, a mAb specific for EBNA1 (a kind gift from Dr Jaap Middeldorp, Free University of Amsterdam, the Netherlands), were used for supershifting. The antibodies were added 15 min before the probe and poly(dI-dC). Transient transfections and luciferase assays. Co-transfections
(b)
were performed at least three times in triplicate in 293A and DG75 cells, using a constant amount of reporter plasmid [0?5 mg pT81lucFR and/or 1 mg p(oriPI/2170Cp)Luc] and varying amounts of expression vector for EBNA1, Oct-1, Oct-2 or Bob.1. pcDNA-3 was added to equalize the amount of DNA in each transfection. bGalactosidase expression from the co-transfected pCMV-b-gal vector was used for normalization of variation in transfection efficiency. Another luciferase vector, RSV-luc, was used as a positive control.
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Fig. 1. Schematic illustration of the oriP region upstream of Cp. (a) Schematic illustration of the region upstream of Cp. Sequence coordinates are from the DNA sequence of the B95-8 EBV genome. The arrow indicates the Cp transcription start site at nt 11336. (b) About 20 repeated motifs, of 30 bases each, constitute the FR. The sequence of several of these repeats is shown, illustrating some of the sequence variation in the FR. Each repeat contains a core EBNA1-binding element (shaded). The motif resembling octamer-binding sites is indicated by an open box or is underlined on the complementary strand. (c) Detailed map of promoter-proximal transcriptional elements present in p(oriPI/”170Cp)Luc. Numbers are positions in relation to the Cp transcription-initiation site (+1). 1262
293A cells were seeded in 6 cm plates and transfected by using FuGENE 6 transfection reagent (Roche) when 75 % confluent. Transfection of DG75 cells was performed with ~86106 cells by using electroporation (960 mF, 280 mV). Luciferase activity was measured in one-fifth of the whole-cell extract at 48 h post-transfection by using a luciferase assay system (Promega). Control transfections using an expression vector for green fluorescent protein showed that approximately 10 % of the DG75 cells were transfected by using this method.
RESULTS AND DISCUSSION In the FR sequence, slightly overlapping the 25 bp fragment that is protected in DNase-protection assays in EBV-positive cells (Rawlins et al., 1985), we found a putative Oct-binding site (Fig. 1b). The Oct family of transcription factors was originally identified as transcriptional regulators when bound to the octamer motif ATGCAAAT. Oct-1 is expressed ubiquitously in all types of tissues (Mu¨ller et al., 1988; Sturm et al., 1988). It interacts with a variety of other Journal of General Virology 86
OCTA
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Two methods were used to demonstrate the interaction of Oct proteins with the FR: EMSA and a luciferase reporter assay. We performed an EMSA to see whether an FR-derived probe (Fig. 3b) could form complexes with proteins extracted from the EBV-positive Burkitt’s lymphoma cell line Rael. The FR probe formed several different protein complexes that were FR-specific (Fig. 2). An excess of unlabelled FR fragment competed efficiently with these complexes (not shown). Complex I (cI), cIII and part of cIV were Oct-specific (Fig. 2, OCTA), which was further confirmed by antibody supershifting. cI was shown to contain Oct-1, whilst cII contained EBNA1 and low amounts of Oct-1 and Oct-2, and cIII and cIV contained Oct-2. Antibodies against the B cell-specific Oct cofactor Bob.1 (also known as OBF-1 and OCA-B) only resulted in a weak reduction in intensity of cI and cII. Several complexes contained more than one protein and, taken together, this suggested a possible interaction between Oct proteins, EBNA1 and the FR in cis. In an attempt to further map the Oct-binding site in the FR, we divided the probe into two parts (Fig. 3b) so that the putative Oct-binding site and the EBNA1-binding site were separated. As suspected, the result showed that EBNA1 and the Oct proteins bound to different parts of the FR probe (Fig. 3a). http://vir.sgmjournals.org
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NE, Part II
To further explore this novel interaction between Oct proteins and the FR, we set up a model system to evaluate its effect on transcription. We constructed a luciferase reporter vector (pT81luc-FR) containing the FR upstream of a thymidine kinase promoter (Fig. 4c). Our results confirmed previous observations (La¨ngle-Rouault et al., 1998) that EBNA1 enhanced luciferase gene expression in 293A cells and showed that our reporter-vector system worked as
NE, FR
transcriptional regulators and activates transcription of small nuclear RNA, histone H2B and immunoglobulin genes (Das & Herr, 1993; Scheidereit et al., 1987; Tanaka et al., 1988). Oct-2 is tissue-specific for neuronal cells and B cells. It is involved in activating transcription from promoters of the immunoglobulin genes (Scheidereit et al., 1987). The primary Oct-2 RNA transcript is subject to alternative splicing, resulting in at least eight different isoforms of the protein (Lillycrop & Latchman, 1992; Liu et al., 1995; Wirth et al., 1991). The splicing pattern differs between B cells and neurons. Oct-2.1 and Oct-2.2 are most prevalent in B cells, whereas Oct-2.4 and Oct-2.5 are the predominant isoforms in neuronal cells. For overexpression of Oct-2, we used a cloned cDNA of Oct-2.6, which is expressed in B cells and has a splicing pattern similar to those of Oct-2.1 and Oct-2.2 (Wirth et al., 1991).
Fig. 2. Oct-1, Oct-2 and EBNA1 form complexes with a probe representing a single repeat of the FR. Nuclear extracts (NE) of latency I Rael cells were used in an EMSA and complexes were supershifted by using specific antibodies. Competition with a cold FR probe (FR) and octamer sequence (OCTA) are shown on the right. Roman numerals to the left indicate the different complexes.
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Fig. 3. EMSA on nuclear extract (NE) from Rael cells. (a) EMSA of a single, full-length repeat of the FR as a probe, or probes consisting of a single repeat of the FR divided into two (Part I and Part II). Note that the Part I and Part II probes are slightly smaller than the control probe in the left-hand lane. (b) Probe of the single repeat of the FR used in the EMSA. The probe was divided into two, Part I and Part II, indicated by an arrow, for the refined binding analysis shown in lanes 2 and 3 in (a). Part I contained the EBNA1-binding site (shaded) and Part II represented a probe containing the putative Oct-binding site (open box). Asterisks indicate bases protected in the DNase-protection assay of Jones et al. (1989). 1263
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(a) Fold activation
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Fig. 4. Oct proteins activate transcription from an FR reporter vector. (a) Luciferase activity obtained in 293A cells cotransfected with pT81luc-FR and increasing amounts (mg) of EBNA1, Oct-2, Oct-1 and/or Bob.1 expression vectors as indicated. Activity is shown as fold activation of the negative-control reporter vector alone (0?5 mg pT81luc-FR; first black bar). Transfections were done in triplicate. Error bars indicate SEM. (b) Luciferase activity obtained in 293A cells transfected with pT81luc-FR and varying amounts (mg) of expression vectors for EBNA1, Oct-2.6, Oct-1 and Bob.1 as indicated. (c) Luciferase reporter vector pT81luc-FR used in the transfection experiments. The FR corresponds to the region between nt 7401 and 8044 in the EBV genome and was inserted upstream of a thymidine kinase promoter in front of the luciferase (luc) gene. Grey bars indicate co-transfection of EBNA1 and an Oct protein.
expected (Fig. 4a). In the same reporter system, we found that overexpression of Oct-2 alone, but not Oct-1, could upregulate the FR-dependent heterologous promoter in 293A cells in the same way as EBNA1 (Fig. 4a). However, Oct-1 could activate transcription together with the B cellspecific co-activator Bob.1 (Fig. 4a). Bob.1 on its own did not induce transcription (data not shown). Thus, EBNA1 enhancement of transcription through the FR could be replaced by Oct proteins. We also detected a small additive effect of co-transfection of EBNA1 with Oct-2 or with Oct-1 plus Bob.1 (Fig. 4b). In the physiological setting, EBNA1 and Oct proteins may cooperate in the regulation of Cp. 293A cells offer a readily transfectable model cell line, although this cellular environment lacks the natural background of EBV infection in B cells. We therefore also carried out transfection of the EBV-negative human B-cell line DG75. EBNA1 activated transcription more efficiently in DG75 than in 293A cells and reached saturation with 0?5 mg EBNA1 vector (data not shown). Oct-1 together with 1264
Bob.1, and Oct-2 alone, both activated transcription through the FR–luciferase reporter system (Fig. 5). Oct-1 also showed a low activation on its own without Bob.1 (data not shown), probably due to the presence of endogenous Bob.1 in B cells. When transfecting DG75 cells with Oct-2, or Bob.1 plus Oct-1, together with EBNA1, we could not detect the additive effect that we saw in the 293A system. This difference in 293A cells can most probably be attributed to the different background of endogenous regulatory factors in B cells, including Oct-2, compared with epithelial cells. Unfortunately, this reporter system could not be used easily in other EBV-carrying B-cell lines, as they showed much lower transfection efficiencies, and endogenous EBV genomes and EBNA1 seemed to interfere with the results in a complex manner. Fig. 5(b) shows the endogenousexpression levels of Oct-1, Oct-2 and Bob.1 in 293A and DG75 cells. Control Western blots of lysates from cells transfected for the luciferase assay showed that the cooperating effect of EBNA1 and the Oct proteins was indeed a result of interaction with the FR sequence (Fig. 5c) Journal of General Virology 86
Oct regulation through EBV oriP
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and not due to any direct effects on the vector-expression system. It clearly showed that there was no effect of cotransfection, either on Oct-2 or on EBNA1 expression levels. To investigate the effect of Oct proteins on the natural EBV promoter, we used another FR–luciferase construct, p(oriPI/2170Cp)Luc, containing parts of the natural Cp of EBV. Vector (1 mg) was transfected into 293A cells together with different amounts of Oct-2.6. Both EBNA1 and Oct-2.6 enhanced promoter activity on their own (Fig. 6). Thus, we demonstrated that Oct transcription factors can functionally interact with the major enhancer in EBV control of transcription in latency, the FR. This is extremely interesting in view of the overwhelming documentation of B cell-specific regulation of EBV latency, involving Cp and
Fig. 6. Oct proteins activate transcription through a Cpcontaining reporter vector. Luciferase activity obtained in 293A cells co-transfected with p(oriPI/”170Cp)Luc and increasing amounts (mg) of EBNA1 and Oct-2.6 expression vector, as indicated, is shown as fold activation of the negative-control reporter vector alone [1 mg p(oriPI/”170Cp)Luc; first black bar]. Transfections were done in triplicate. Error bars indicate SEM. http://vir.sgmjournals.org
2
Fig. 5. (a) Luciferase activity obtained in DG75 cells transfected with pT81luc-FR and varying amounts (mg) of the expression vectors for EBNA1, Oct-1, Bob.1 and Oct2.6 as indicated. Error bars indicate SEM. (b) Western blot analysis comparing protein levels of Oct-2, Bob.1 and Oct-1 in 293A and DG75 cells. The membrane was probed with protein-specific antibodies as indicated on the left. (c) Western blot analysis of DG75 cells transfected with varying amounts (mg) of expression vectors for EBNA1 and Oct-2 as indicated. An increase in the amount of Oct-2 vector added did not affect the amount of EBNA1 expressed and EBNA1 did not affect Oct-2 expression. Proteins were identified by using specific antibodies as indicated on the left. Grey bars indicate co-transfection of EBNA1 and an Oct protein.
cellular and EBV-specific transcription factors (Nilsson et al., 1993; Sung et al., 1991). Oct proteins may be involved in both activation and repression. In certain binding conformations, Oct factors cooperate with activating co-regulators, such as Bob.1, but there is also considerable evidence that Oct can recruit inhibitory molecules; for example, the neuronal forms of Oct-2 can repress the tyrosine hydroxylase gene promoter (Dawson et al., 1994) and Oct-2 represses the herpes simplex virus (HSV) immediate-early promoter in neurons (Lillycrop et al., 1991). This potentially enables Octdependent regulation to be involved in the switches between different forms of latency. The sequence of the FR contains imperfect consensus octamer-binding sites. However, it is already wellestablished that Oct transcription factors can interact with DNA in several different configurations and with binding motifs that diverge extensively from the so-called octamer consensus motif (ATGCAAAT) that is found in all promoter enhancers within the IgH locus. One example is the TAATGARAT motif in HSV (Lillycrop & Latchman, 1992) and another is the more recently discovered MORE and PORE sequences that can either exclude or include Bob.1 in the complex (Tomilin et al., 2000). In the FR, there are three interspersed ATATAAAT motifs that best match the consensus octamer. Oct proteins and EBNA1 showed a preference for binding by themselves to the FR probe, although we also found some indication of combined binding in one of the EMSA complexes (cII). Binding of Oct and EBNA1 was mapped to different ends of the FR repeat. The results of a DNaseprotection assay (Jones et al., 1989), together with our results from the EMSA, suggest that there may be some 1265
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steric hindrance in binding both EBNA1 and Oct protein to a single FR repeat. Despite this, there is still the possibility that EBNA1 binds to one FR repeat and the Oct protein to another. As shown by Ambinder et al. (1990), the affinity of the EBNA1–DNA interaction, and presumably also the Oct–DNA interaction, varies detectably with the small sequence variations detected in the FR motifs. It seems that EBNA1 and Oct proteins can both bind together to longer probes with two repeats, but the number of complexes then also increases and complicates the analysis (data not shown). It is interesting to note that the Oct-binding motifs in the FR that mostly resemble the consensus Oct site are in fact adjacent to variant EBNA1 motifs with reduced binding affinity for EBNA1. The relative concentrations of the proteins can also play a role in vivo. EBNA1 expression is relatively low in latency I cell lines and the EBNA1 signal may be drowned by higher levels of Oct proteins. In vivo, the configuration of the entire DNA region from the FR to Cp and the multimerization of EBNA1 following DNA binding is likely to confer additional conditions for recruitment of transcription factors and their co-factors.
Das, G. & Herr, W. (1993). Enhanced activation of the human histone
H2B promoter by an Oct-1 variant generated by alternative splicing. J Biol Chem 268, 25026–25032. Dawson, S. J., Yoon, S. O., Chikaraishi, D. M., Lillycrop, K. A. & Latchman, D. S. (1994). The Oct-2 transcription factor represses
tyrosine hydroxylase expression via a heptamer TAATGARAT-like motif in the gene promoter. Nucleic Acids Res 22, 1023–1028. Ernberg, I., Falk, K., Minarovits, J., Busson, P., Tursz, T., Masucci, M. G. & Klein, G. (1989). The role of methylation in the phenotype-
dependent modulation of Epstein–Barr nuclear antigen 2 and latent membrane protein genes in cells latently infected with Epstein–Barr virus. J Gen Virol 70, 2989–3002. Gahn, T. A. & Sugden, B. (1995). An EBNA-1-dependent enhancer
acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene. J Virol 69, 2633–2636. Jin, X. W. & Speck, S. H. (1992). Identification of critical cis ele-
ments involved in mediating Epstein-Barr virus nuclear antigen 2-dependent activity of an enhancer located upstream of the viral BamHI C promoter. J Virol 66, 2846–2852. Jones, C. H., Hayward, S. D. & Rawlins, D. R. (1989). Interaction
of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites. J Virol 63, 101–110. Klein, G., Dombos, L. & Gothoskar, B. (1972). Sensitivity of
Epstein–Barr virus (EBV) producer and non-producer human lymphoblastoid cell lines to superinfection with EB-virus. Int J Cancer 10, 44–57.
Although the in vivo situation is more complex than our model, we propose that the biological significance of our findings relates to the B cell-specific regulation of viral latency depending on Cp.
Kupfer, S. R. & Summers, W. C. (1990). Identification of a
ACKNOWLEDGEMENTS
La¨ngle-Rouault, F., Patzel, V., Benavente, A., Taillez, M., Silvestre, N., Bompard, A., Sczakiel, G., Jacobs, E. & Rittner, K. (1998). Up to
J. A. was a recipient of a fellowship from the Swedish Foundation for Strategic Research (SSF), Infection and Vaccinology PhD programme. This work was also supported by the Swedish Cancer Society, the Swedish Research Council, the Swedish Children Cancer Society and the Board for Internationalization of Research (STINT). S. P. is supported by the SSF and the Swedish Cancer Society. We are grateful for the technical assistance of Anita Westman and to Stephen Malin (MTC, now at The University of Vienna, Austria) for valuable comments. The EBNA1 expression vector was kindly provided by Professor Maria Masucci, MTC, Karolinska Institutet, Sweden.
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