GSFForschungszentrum für Umwelt und Gesundheit, Institut für Klinische Molekularbiologie und Tumorgenetik, Marchioninistr. 25, D-81377 München, Germany1
Deutsches Krebsforschungszentrum (DKFZ), Forschungsschwerpunkt Angewandte Tumorvirologie, D-69120 Heidelberg, Germany2
Author for correspondence: G. Laux. Fax +49 89 7099500. e-mail laux{at}gsf.de
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Abstract |
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BL is frequently associated with EpsteinBarr virus (EBV), a lymphotropic -herpesvirus. Infection of primary B cells in vitro with EBV results in cell transformation and the establishment of lymphoblastoid cell lines (LCL) that express six viral latent nuclear antigens (EBNAs) and three latent membrane proteins (LMPs). In BL cells in vivo, the expression of EBV latent proteins is restricted to EBNA1 (Rickinson & Kieff, 1996
). The two different programmes of EBV latent gene expression are termed EBV latency type I in BL and type III in LCL (Rowe et al., 1987
). Different promoters regulate EBNA1 expression in latency I and III. EBNA1 is expressed from the Qp promoter (Schaefer et al., 1995
) in BL whereas, in LCL, all EBNA genes are expressed from the Cp promoter. The individual mRNAs are generated by alternative splicing of long primary transcripts (Kieff, 1996
).
Immortalization of B cells by EBV is assumed to be an important step during the pathogenesis of BL. Therefore, an intriguing question is which factors implement the restricted EBV gene expression programme found in BL; i.e. which factors induce the switch from EBV latency type III to type I. Several regulatory factors of the EBNA1 promoter Qp, active in BL cells, have been identified so far, among them viral proteins (EBNA1), immune response-dependent factors (IRFs, STATs) and cell-cycle regulating proteins (E2F, Rb) (Chen et al., 1999 ; Davenport & Pagano, 1999
; Nonkwelo et al., 1997
; Ruf & Sample, 1999
; Schaefer et al., 1997b
; Zhang & Pagano, 1997
, 1999
). Methylation of the EBV genome has been demonstrated to be a prerequisite for the maintenance of latency type I by inhibiting the activation of the Cp promoter, which is active in LCL (Robertson, 2000
). Until now, a latency III-to-I switch in B cells has not been observed in vitro.
In order to investigate the genetic events that contribute to the pathogenesis of BL, we overexpressed the proto-oncogene myc in the conditional LCL EREB2-5 (Pajic et al., 2000 ; Polack et al., 1996
). This cell line carries the EBNA2 deletion mutant P3HR-1 and an expression plasmid for an EBNA2oestrogen receptor fusion protein, which renders EBNA2 function and EBV immortalization dependent on the presence of oestrogen (Kempkes et al., 1995
). Ectopic overexpression of myc in EREB2-5 cells allows the establishment of cell lines that proliferate independently of EBNA2 and express features typical of BL cells (in vivo phenotype) (Polack et al., 1996
). Here, we asked whether the observed switch from an LCL phenotype to a BL phenotype was accompanied by an EBV latency III-to-I switch. The activities of the EBNA promoters Qp and Cp were analysed in the cell lines A1, in which myc is overexpressed under the control of Ig
enhancer elements (Polack et al., 1996
), and P493-6, in which myc expression can be controlled by a tetracycline-responsive promoter (Pajic et al., 2000
).
Qp- and Cp-initiated EBNA1 transcripts can be detected and distinguished by RTPCR (Schaefer et al., 1996 ; Schlager et al., 1996
). Reverse transcription was performed with 5·0 µg total RNA, 0·5 µg oligo(dT) primer, 0·5 mM dNTPs, 200 U reverse transcriptase (Superscript II, Gibco BRL), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 50 mM TrisHCl (pH 8·3) and 20 U RNase inhibitor (RNasin) in 20 µl at 42 °C for 50 min. The reaction was stopped by heating at 70 °C for 15 min.
A 1/50 aliquot of the RT reaction was subjected to 25 cycles of PCR. For amplification of Qp-initiated transcripts, PCR was performed with 5 pmol primer, 0·1 mM dNTPs, 2·5 U Taq DNA polymerase, 1 mM MgCl2, 0·1% Triton X-100 and 10 mM TrisHCl (pH 9·0) in 50 µl. Each thermocycle was composed of 1 min denaturation at 94 °C, 30 s primer hybridization at 58 °C and 1 min synthesis at 72 °C. For amplification of Cp-initiated transcripts, PCR was performed with 10 pmol primer, 0·2 mM dNTPs and 5 U Taq DNA polymerase (Promega) in 1·5 mM MgCl2, 0·1% Triton X-100 and 10 mM TrisHCl (pH 9·0). The PCR programme was as above except that the primer hybridization and DNA synthesis phases were extended to 1 and 3 min, respectively.
PCR products were resolved by agarose gel electrophoresis and subjected to Southern blotting according to standard protocols (Sambrook et al., 1989 ). Blots were hybridized with a 32P-labelled U-exon probe. Radiolabelling of DNA was performed by random-primed labelling according to the protocols of the manufacturer.
A QK primer pair was applied for the detection of Qp activity in A1 and P493-6 cells (Schaefer et al., 1995 ). During the lytic cycle, the Fp promoter generates transcripts that overlap Qp-initiated transcripts and may mimic Qp activity in RTPCR analysis (Fig. 1
). Therefore, we controlled for the presence of lytic, Fp-initiated EBNA1 transcripts by a separate PCR with an FK primer pair. Neither Qp- nor Fp-initiated EBNA1 transcripts were present in A1 and P493-6 cells. In control cells, these transcripts could be detected easily (Fig. 2
). In order to exclude the possibility that the negative results in our cellular system were due to point mutations in the primer-binding sites, we sequenced the respective regions in A1 cells. No mutations were found in the sequences covering the F, Q and K primer-binding sites (data not shown).
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In LCL, Cp-initiated transcripts are spliced to generate the mRNA of all EBNA genes (Rickinson & Kieff, 1996 ). We asked whether the observed Cp activity in A1 and P493-6 cells would also result in the expression of other EBNAs and analysed the expression of EBNA3 transcripts (EBNA3A, B and C). PCR was performed with a 5' primer in the W2 exon that is common for all EBNA3 transcripts and 3' primers in the unique EBNA3A, B or C coding regions. In EREB2-5, A1 and P493-6 cells, transcripts of EBNA3A, B and C were detected. The results of the EBNA3C analysis are shown in Fig. 3
as a representative of all EBNA3 genes.
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Kerr et al. (1992) studied somatic cell hybrids and observed a downregulation of Wp/Cp and an upregulation of Qp in hybrid cell lines that had lost the LCL phenotype and acquired the phenotype of the non-B cell fusion partners. However, all cell hybrids that retained the LCL-like phenotype also kept the EBV latency III programme. This was always the case when a BL cell line was fused with an LCL, showing that the LCL phenotype was dominant over the BL phenotype (Wolf et al., 1993
).
In contrast, our system allows the switching from an LCL to a BL phenotype in the same cells. Similar phenotypic changes were observed after the transition of Jijoye BL cells to the P3HR-1 daughter cell line. Jijoye cells show an LCL phenotype, including the EBV latency III transcription programme, whereas P3HR-1 cells have acquired an EBNA2 deletion and have lost the expression of typical LCL surface antigens. EBNA1 is expressed exclusively from Wp in P3HR-1 cells. This is in contrast to the cell system used here. However, the factors that determine the use of Wp rather than Cp are unknown in P3HR-1 cells. It is also unclear whether there is a difference in virus promoter regulation if EBNA2 is expressed from an episome in trans, as in our cellular system (Kempkes et al., 1996 ), or from the same genome in cis. This can be overcome by generating recombinant EBV with a conditional EBNA2 based on the EBV B95-8 strain (Delecluse et al., 1998
, 1999
).
In another analysis, cells of an EBV-negative BL line were infected with the P3HR-1 virus (Schlager et al., 1996 ). The activities of the EBNA promoters were monitored during a time-course of several hours after infection. It was observed that both Qp and Cp were activated. These data suggested that the environment of a BL cell might support the activity of both EBV latency I (Qp) and III (Cp) promoters and that the P3HR-1 EBV strain does not contain defective Qp or Cp promoters.
We intended to construct an EBV latency III-to-I switch in vitro by overexpressing myc and inactivating EBNA2 in parallel. It is striking that the EBNA transcription programme characteristic of LCL remained active despite the enormous phenotypic changes. Repression of Wp and Cp by methylation has been demonstrated to be a prerequisite for Qp activation in B cells (Schaefer et al., 1997 a). It is likely that the EBV genome is not methylated in the same way in A1 and P493-6 cells as in BL cells. At present it is not clear which factors trigger methylation of EBV promoters in B cells. Our data imply that the overexpression of myc and inactivation of EBNA2 are not sufficient for Wp/Cp methylation.
It has been reported that elevated levels of EBNA1 can inhibit Qp activity (Sample et al., 1992 ). A1 and P493-6 cells expressed higher levels of EBNA1 protein than did EREB2-5 cells. However, the levels of EBNA1 protein in A1 and P493-6 cells were comparable with levels found in BL phenotype I cells Rael and MutuI. Additionally, MutuIII cells, representing Mutu cells in EBV latency III, expressed levels of EBNA1 protein comparable to those of EREB2-5 cells (data not shown). These data suggest that the higher EBNA1 protein levels in A1 and P493-6 cells may not be inhibitory for Qp activity.
EBNA3A, -B and -C transcripts were also detected in A1 and P493-6 cells. However, the presence of EBNA3 proteins could not be verified, due to the lack of antibodies that recognize EBNA3 proteins of the EBV type 2 P3HR-1 strain specifically. Members of the EBNA3 protein family have been demonstrated to modulate EBNA2-dependent transcription and are also likely to contribute to transcriptional regulation in the absence of EBNA2 (Cludts & Farrell, 1998 ; Marshall & Sample, 1995
). It remains unclear whether EBNA3 proteins could contribute to the activation of Cp in A1 and P493-6 cells. The answer to these questions could be provided by establishing a similar cell system based on a B95-8 recombinant EBV, for which the appropriate reagents are available.
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Acknowledgments |
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Footnotes |
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c Present address: Universität Köln, Institut für Physiologie, Robert-Koch-Str. 39, D-50931 Köln, Germany.
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References |
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Cludts, I. & Farrell, P. J. (1998). Multiple functions within the EpsteinBarr virus EBNA-3A protein. Journal of Virology 72, 1862-1869.
Cutrona, G., Ulivi, M., Fais, F., Roncella, S. & Ferrarini, M. (1995). Transfection of the c-myc oncogene into normal EpsteinBarr virus-harboring B cells results in new phenotypic and functional features resembling those of Burkitt lymphoma cells and normal centroblasts. Journal of Experimental Medicine 181, 699-711.[Abstract]
Davenport, M. G. & Pagano, J. S. (1999). Expression of EBNA-1 mRNA is regulated by cell cycle during EpsteinBarr virus type I latency. Journal of Virology 73, 3154-3161.
Delecluse, H. J., Hilsendegen, T., Pich, D., Zeidler, R. & Hammerschmidt, W. (1998). Propagation and recovery of intact, infectious EpsteinBarr virus from prokaryotic to human cells. Proceedings of the National Academy of Sciences, USA 95, 8245-8250.
Delecluse, H. J., Pich, D., Hilsendegen, T., Baum, C. & Hammerschmidt, W. (1999). A first-generation packaging cell line for EpsteinBarr virus-derived vectors. Proceedings of the National Academy of Sciences, USA 96, 5188-5193.
Evans, T. J., Farrell, P. J. & Swaminathan, S. (1996). Molecular genetic analysis of EpsteinBarr virus Cp promoter function. Journal of Virology 70, 1695-1705.[Abstract]
Henriksson, M. & Lüscher, B. (1996). Proteins of the Myc network: essential regulators of cell growth and differentiation. Advances in Cancer Research 68, 109-182.[Medline]
Hotchin, N. A., Allday, M. J. & Crawford, D. H. (1990). Deregulated c-myc expression in EpsteinBarr-virus-immortalized B-cells induces altered growth properties and surface phenotype but not tumorigenicity. International Journal of Cancer 45, 566-571.
Kempkes, B., Spitkovsky, D., Jansen-Durr, P., Ellwart, J. W., Kremmer, E., Delecluse, H. J., Rottenberger, C., Bornkamm, G. W. & Hammerschmidt, W. (1995). B-cell proliferation and induction of early G1-regulating proteins by EpsteinBarr virus mutants conditional for EBNA2. EMBO Journal 14, 88-96.[Abstract]
Kempkes, B., Zimber-Strobl, U., Eissner, G., Pawlita, M., Falk, M., Hammerschmidt, W. & Bornkamm, G. W. (1996). EpsteinBarr virus nuclear antigen 2 (EBNA2)oestrogen receptor fusion proteins complement the EBNA2-deficient EpsteinBarr virus strain P3HR1 in transformation of primary B cells but suppress growth of human B cell lymphoma lines. Journal of General Virology 77, 227-237.[Abstract]
Kerr, B. M., Lear, A. L., Rowe, M., Croom-Carter, D., Young, L. S., Rookes, S. M., Gallimore, P. H. & Rickinson, A. B. (1992). Three transcriptionally distinct forms of EpsteinBarr virus latency in somatic cell hybrids: cell phenotype dependence of virus promoter usage. Virology 187, 189-201.[Medline]
Kieff, E. (1996). EpsteinBarr virus and its replication. In Fields Virology , pp. 2343-2396. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:LippincottRaven.
Lombardi, L., Newcomb, E. W. & Dalla-Favera, R. (1987). Pathogenesis of Burkitt lymphoma: expression of an activated c-myc oncogene causes the tumorigenic conversion of EBV-infected human B lymphoblasts. Cell 49, 161-170.[Medline]
Marshall, D. & Sample, C. (1995). EpsteinBarr virus nuclear antigen 3C is a transcriptional regulator. Journal of Virology 69, 3624-3630.[Abstract]
Nesbit, C. E., Tersak, J. M. & Prochownik, E. V. (1999). MYC oncogenes and human neoplastic disease. Oncogene 18, 3004-3016.[Medline]
Nonkwelo, C., Ruf, I. K. & Sample, J. (1997). Interferon-independent and -induced regulation of EpsteinBarr virus EBNA-1 gene transcription in Burkitt lymphoma. Journal of Virology 71, 6887-6897.[Abstract]
Pajic, A., Spitkovsky, D., Christoph, B., Kempkes, B., Schuhmacher, M., Staege, M. S., Brielmeier, M., Ellwart, J., Kohlhuber, F., Bornkamm, G. W., Polack, A. & Eick, D. (2000). Cell cycle activation by c-myc in a burkitt lymphoma model cell line. International Journal of Cancer 87, 787-793.
Polack, A., Hortnagel, K., Pajic, A., Christoph, B., Baier, B., Falk, M., Mautner, J., Geltinger, C., Bornkamm, G. W. & Kempkes, B. (1996). c-myc activation renders proliferation of EpsteinBarr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1. Proceedings of the National Academy of Sciences, USA 93, 10411-10416.
Rickinson, A. B. & Kieff, E. (1996). EpsteinBarr virus. In Fields Virology , pp. 2397-2446. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia:LippincottRaven.
Robertson, K. D. (2000). The role of DNA methylation in modulating EpsteinBarr virus gene expression. Current Topics in Microbiology and Immunology 249, 21-34.[Medline]
Rowe, M., Rowe, D. T., Gregory, C. D., Young, L. S., Farrell, P. J., Rupani, H. & Rickinson, A. B. (1987). Differences in B cell growth phenotype reflect novel patterns of EpsteinBarr virus latent gene expression in Burkitts lymphoma cells. EMBO Journal 6, 2743-2751.[Abstract]
Ruf, I. K. & Sample, J. (1999). Repression of EpsteinBarr virus EBNA-1 gene transcription by pRb during restricted latency. Journal of Virology 73, 7943-7951.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sample, J., Henson, E. B. & Sample, C. (1992). The EpsteinBarr virus nuclear protein 1 promoter active in type I latency is autoregulated. Journal of Virology 66, 4654-4661.[Abstract]
Schaefer, B. C., Strominger, J. L. & Speck, S. H. (1995). Redefining the EpsteinBarr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines. Proceedings of the National Academy of Sciences, USA 92, 10565-10569.[Abstract]
Schaefer, B. C., Strominger, J. L. & Speck, S. H. (1996). A simple reverse transcriptase PCR assay to distinguish EBNA1 gene transcripts associated with type I and II latency from those arising during induction of the viral lytic cycle. Journal of Virology 70, 8204-8208.[Abstract]
Schaefer, B. C., Strominger, J. L. & Speck, S. H. (1997a). Host-cell-determined methylation of specific EpsteinBarr virus promoters regulates the choice between distinct viral latency programs. Molecular and Cellular Biology 17, 364-377.[Abstract]
Schaefer, B. C., Paulson, E., Strominger, J. L. & Speck, S. H. (1997b). Constitutive activation of EpsteinBarr virus (EBV) nuclear antigen 1 gene transcription by IRF1 and IRF2 during restricted EBV latency. Molecular and Cellular Biology 17, 873-886.[Abstract]
Schlager, S., Speck, S. H. & Woisetschlager, M. (1996). Transcription of the EpsteinBarr virus nuclear antigen 1 (EBNA1) gene occurs before induction of the BCR2 (Cp) EBNA gene promoter during the initial stages of infection in B cells. Journal of Virology 70, 3561-3570.[Abstract]
Wolf, J., Pawlita, M., Klevenz, B., Frech, B., Freese, U. K., Muller-Lantzsch, N., Diehl, V. & zur Hausen, H. (1993). Down-regulation of integrated EpsteinBarr virus nuclear antigen 1 and 2 genes in a Burkitt lymphoma cell line after somatic cell fusion with autologous EBV-immortalized lymphoblastoid cells. International Journal of Cancer 53, 621-627.
Zhang, L. & Pagano, J. S. (1997). IRF-7, a new interferon regulatory factor associated with EpsteinBarr virus latency. Molecular and Cellular Biology 17, 5748-5757.[Abstract]
Zhang, L. & Pagano, J. S. (1999). Interferon regulatory factor 2 represses the EpsteinBarr virus BamHI Q latency promoter in type III latency. Molecular and Cellular Biology 19, 3216-3223.
Received 26 March 2001;
accepted 9 August 2001.