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Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at Göteborg University, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden
Correspondence
Lars Rymo
lars.rymo{at}clinchem.gu.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number of the sequence reported in this paper is EF164992.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The EBV-encoded latent membrane protein 1 (LMP1) is critically involved in the immortalization and proliferation of B-cells latently infected by EBV (Kaye et al., 1993, 1995; Kilger et al., 1998). It also has the ability to transform rodent fibroblast and human cell lines in culture (Baichwal & Sugden, 1988; Fahraeus et al., 1990b; Moorthy & Thorley-Lawson, 1993; Wang et al., 1985). LMP1 is expressed in latency type II and III. LMP1 transcription can be initiated from two promoters in the EBV genome, a proximal promoter referred to as the ED-L1 promoter and a distal promoter mapped to the terminal repeats named LT-R1 (Chang et al., 1997; Fennewald et al., 1984; Hudson et al., 1985; Sadler & Raab-Traub, 1995). The viral EBV nuclear antigen 2 (EBNA2) protein is the most potent transactivator of the LMP1 gene through the ED-L1 promoter, but requires other viral and cellular genes for transactivation (Kieff & Rickinson, 2001). In latency type III, LMP1 expression is EBNA2-dependent, while in latency II it is EBNA2-independent. In latency II, LMP1 expression can be driven by both the LT-R1 and the ED-L1 promoters (Sadler & Raab-Traub, 1995).
Several regulatory elements in the LMP1 ED-L1 promoter region have been shown to mediate both transcription repression and activation. The RBP-J
sites, a PU-box and an AP-2 consensus site confer activation of the promoter in an EBNA2-dependent manner. Other elements such as an Sp factor-binding site, cyclic AMP response element (CRE), E-box element, octamer-binding site and interferon-stimulated response element (ISRE) have been described to be involved in the regulation of the promoter in an EBNA2-independent manner (reviewed by Zetterberg & Rymo, 2005). These studies have been carried out on the prototype B95-8 strain of EBV, which originates from a marmoset cell line infected with EBV from an IM patient (Miller et al., 1972). However, sequence variations in the lmp1 gene have been reported for different EBV strains, both in the coding sequence and in the promoter region (Chen et al., 1992; Hu et al., 1993; Sandvej et al., 2000; Takacs et al., 2001; Zhou et al., 2001). It is thus necessary to consider the effect of sequence variations in promoter regulation.
In this study, the LMP1 promoter in the EREB2.5-derived virus was sequenced to monitor possible differences between this strain and the B95-8 strain that may affect promoter regulation. The EREB2.5 cell line is a useful tool in the study of LMP1 as it is conditional for the activation of EBNA2, with the consequence that the LMP1 promoter can be switched on or off by the addition or removal of 
-oestradiol from the medium (Kempkes et al., 1995). The LMP1 sequence in EREB2.5 originates from the P3HR1 viral strain (Kempkes et al., 1995). Sequencing of the LMP1 regulatory sequence (LRS), defined here as positions –634 to +40 relative to transcription start site (+1) in the P3HR1 virus, revealed 25 nt substitutions and one insertion when compared with the B95-8 sequence. One of the substitutions, a C to an A at position –43 relative to the transcription initiation site, was within the CRE element. Notably, sequence variations in this CRE site have also been found in several other EBV strains (Chen et al., 1992; Hu et al., 1993; Sandvej et al., 2000; Takacs et al., 2001; Zhou et al., 2001). We have previously shown that the CRE element plays a significant role in LMP1 activation, both in an EBNA2-dependent and -independent manner (Sjoblom et al., 1998). Here, we investigated the effect of CRE-sequence variants present in different EBV strains on the ability of the CRE-binding transcription factors to interact with the site in order to elucidate the functional consequences for the regulation of the LMP1 promoter.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A6 and control vector (pSV2gpt) have been described earlier (Ricksten et al., 1987). The LRS is defined as nt 169 019–169 692 of B95-8 EBV DNA (GenBank accession no. AJ507799
[GenBank]
), which corresponds to positions –634 to +40 relative to the transcription initiation site. The B95-8 LRS and B95-8 LRS (CREMUT) luciferase reporter plasmids were generated by transferring HindIII digested LRS fragments from the corresponding plasmids in the CAT reporter system (Fahraeus et al., 1990a; Sjoblom et al., 1998) into the HindIII site in the pGL3-Basic vector (Promega). The P3HR1 LRS was created by PCR-amplification of DNA purified from EREB2.5 cells, using primers that correspond to positions –634 and +40 relative to the transcription initiation site of the LMP1 promoter, along with a HindIII restriction site. The PCR product was digested and cloned into the HindIII site in the pGL3-Basic vector. The B95-8 LRS (CREP3HR1) was created by site-directed mutagenesis in the B95-8 LRS plasmid, using an oligonucleotide containing the C to A substitution at position –43 in LRS. All constructs were verified by dideoxy sequencing utilizing the ABI Prism 310 automated sequencer (Applied Biosystems) and standard protocols. Sequencing of the LRS in P3HR1 was carried out on DNA prepared from EREB2.5 cells with a GenoM-48 robot (GenoVision AS).
Cell culture, DNA transfections and reporter gene assays.
EREB2.5 is a transformed lymphoblastoid cell line expressing conditional EBNA2 (ER-EBNA2) and its activity is regulated by oestrogen (Kempkes et al., 1995). Jijoye is an EBV-positive BL cell line (Pulvertaft, 1965). WW1-LCL is an EBV-immortalized LCL cell line (Gregory et al., 1988) and DG75 is an EBV-negative BL cell line (Ben-Bassat et al., 1977). The cells were maintained as suspension cultures in RPMI 1640 medium supplemented with 10 % fetal calf serum (Life Technologies), 100 U penicillin ml–1 and 100 µg streptomycin ml–1. The EREB2.5 cell line was also supplemented with 1 µM -oestradiol (-oestradiol-water soluble; Sigma). Transient transfections were carried out by electroporation using 5x106 cells and 10 µg reporter plasmids as described previously (Sjoblom et al., 1998). Co-transfections with 2.7 nmol DNA of the EBNA2 expression vector (pEA6) or the empty vector (pSV2gpt) were done in DG75 cells. Cells were harvested after 48 h and assayed for luciferase activity using the Luciferase Assay System (Promega) according to the manufacturer's instructions. A TD 20/20 luminometer (Turner Design Instrument) was used for the detection of luciferase activity. In addition, half the cells harvested after transfection were used for immunoblot analysis in parallel to reporter assay.
Electrophoretic mobility-shift assays (EMSAs).
Nuclear extract preparation and the EMSA-binding reactions were carried out as described previously (Sjoblom et al., 1998). All oligonucleotides were purchased from Invitrogen. EMSAs were carried out using double-stranded synthetic [
-32P]ATP-labelled oligonucleotides corresponding to LRS –50 to –19 position from B95-8, as well as the specific sequence variations in the CRE site detected in P3HR1, Rael, Raji or NPC with blunt ends as shown in Fig. 2. A mutation introduced in the Sp1 site of all probes eliminated the binding of Sp1 factors and facilitated the interpretation of the factor-binding patterns (Sjoblom et al., 1998). The competing oligonucleotides were added at an excess before the addition of the labelled probe and samples were incubated for 20 min at room temperature. In supershift experiments, after the 20 min incubation of nuclear extract with probe, 3–4 µl of the antibody was added and incubated for another 60 min at 4 °C. The antibodies CREB1 (sc-186x), CREB1 (sc-25785x), ATF1 (sc-270x), ATF2 (sc-187x), c-Jun (sc-1694x) and CREB2 (sc-200x) were purchased from Santa Cruz. The samples were separated by electrophoresis in 5 % polyacrylamide gels (acrylamide : bisacrylamide, 29 : 1) in 0.5x TBE for 3 h at 300 V. The bands were either visualized by autoradiography or exposed to a phosphoimage screen and scanned by a Typhoon 9200.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). A ‘C’ to ‘A’ substitution was located in the CRE factor-binding site present in the LRS. The rest of the sequence differences between the two viral strains were located outside the previously reported transcription regulatory elements (Fig. 1a). Variations reported in the CRE site of several other EBV strains are depicted in Fig. 1(b). The EBV strains CAO and C15 are NPC-derived virus isolates that contain a 2 bp substitution in the CRE site (Chen et al., 1992; Hu et al., 1993), and they have an Asian origin. This variation has also been reported for virus isolates from several HD, IM and asymptomatic carriers referred to as group D (Sandvej et al., 2000; Zhou et al., 2001). The Rael and Raji viral strains contain a C to G substitution in the same position as the C to A substitution in the CRE site in P3HR1 (Takacs et al., 2001). The P3HR1 cell line is a subline of the Jijoye BL cell line and has an African origin like the Raji and Rael cell lines (Epstein et al., 1966; Hinuma et al., 1967; Klein et al., 1972; Pulvertaft, 1965). Notably, the phylogenetic tree based on the LMP1 sequence constructed by Kanai et al. (2007) showed that the Asian EBV isolates contain large differences compared with the African BL isolates suggesting independent selection for the CRE-site sequence variants. Stable, genetic variations in CRE sites have also been described for several other promoters (Mitchison, 2001). It has been hypothesized that the activity of some cis-elements such as the CRE site can be changed to a lower level by subtle sequence variations that modulate the level of expression of genes important for cell survival (Mitchison, 2001). The present study together with the reports mentioned above support the notion that the CRE element in the LRS is a preferred target for such mutations that in a certain cellular context might confer evolutionary advantages to the EBV-infected cell and are therefore conserved.
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). In the present study, we compared the factor-binding properties of the B95-8 and P3HR1 LRS CRE sites using nuclear extracts from the EBV-negative DG75 line and B95-8 and P3HR1-derived LRS (–50/–19) oligonucleotides as probes. Five specific complexes bound to the P3HR1-derived probe. The complexes represented by bands I and II corresponded to factors binding outside the CRE site, since the bands were competed away by a probe with a mutated CRE site (Fig. 2a
, lane 5). The three CRE-specific complexes represented by bands III, IV and V were detected with both the B95-8- and the P3HR1-derived probes. The binding efficiencies of the different CRE complexes, however, appeared to differ (Fig. 2a). The CRE-binding complex III dominated in the factor-binding pattern obtained with the P3HR1-derived probe, whereas complexes IV and V dominated in the pattern obtained with the B95-8-derived probe. Supershift experiments with anti-ATF1, anti-CREB1, anti-ATF2 and anti-c-Jun antibodies, respectively, were carried out to confirm the identity of the factors binding to the CRE site in the P3HR1-derived LRS CRE probe (Fig. 2b). ATF1 and CREB1 antibodies shifted complexes IV and V confirming our previous study (Sjoblom et al., 1998). However, antibodies against ATF2 and c-Jun did not shift complex III in the P3HR1-derived probe and only weakly diminished this complex in the B95-8-derived probe (Fig. 2b). Thus, our conclusions in the previous study were not verified on this point (Sjoblom et al., 1998). Furthermore, EMSA competition experiments using CRE sequences known to bind the different members of the CRE-binding transcription factor family with different affinities, confirmed that complex III did not represent binding of c-Jun–ATF2 (unpublished data). Thus, the identity of the CRE-binding factors in complex III remains to be elucidated. It was also noted that this complex was weak or not detected with WW1-LCL nuclear extract (Fig. 2d, e). To assess the difference in protein-binding efficiency of the different transcription factor complexes with the CRE site in a more quantitative way, EMSA competition experiments were performed with DG75 nuclear extracts and a 13-, 25- or 50-fold excess of unlabelled LRS CRE probe from B95-8 and P3HR1, respectively (Fig. 2c). The relative efficiency of binding was calculated as the ratio between the intensity of given EMSA bands obtained with one competitor and that of the corresponding bands without competitor. The C to A substitution in the P3HR1 CRE site led to an approximately twofold higher-binding efficiency of the factors in complex III and an approximately twofold lower-binding efficiency of the ATF1–CREB1 heterodimer. To confirm the binding of the same factors to both the B95-8 and P3HR1 CRE site in an EBV-positive context, EMSA was performed with WW1-LCL nuclear extract (Fig. 2d). A similar binding pattern was observed between DG75 and WW1-LCL extracts for both probes, although complex III appeared to be weaker in WW1-LCL in both cases (Fig. 2d). In addition, EMSAs were performed with WW1-LCL nuclear extract and probes encompassing the corresponding LRS CRE sequences of B95-8, P3HR1, Raji/Rael and NPC viral strains, respectively (Fig. 2e). The EMSA bands corresponding to the positions of the ATF1–CREB1 complexes were weak or not detectable with the LRS CRE site in the Raji/Rael- and NPC-derived probes (Fig. 2e). EMSA supershift experiments using an antibody reactive against ATF1, CREB1 and CREM (the anti-CREB1 antibody) shifted the ATF1–CREB1 complexes obtained with probes carrying the B95-8- and P3HR1-derived LRS CRE sites and the CRE-consensus sequence as expected confirming the identity of the ATF1–CREB1 complexes when using the WW1-LCL nuclear extract (Fig. 2e, lanes 2 and 5). However, the antibody did not shift any of the weak bands in the ATF1–CREB1 heterodimer positions formed with probes containing the corresponding LRS CRE site representing Raji, Rael or the NPC C15 and CAO. This indicated that no such complex had been formed, presumably because the LRS CRE sequence in these EBV lines has a considerably reduced ability to bind the ATF1–CREB1 factors (Fig. 2e). This notion was strengthened by the results of EMSA experiments with unlabelled oligonucleotides containing LRS CRE-sequence variants representing Raji, Rael, NPC C15 and CAO virus isolates as competitors against the B95-8-derived probe. Even in a large excess, the variant oligonucleotides did not compete out the ATF1–CREB1 heterodimer complexes (data not shown).
ATF1 and CREB1 contain phosphorylation-dependent activation domains and become transcriptional activators when phosphorylated at specific serine or threonine residues (Livingstone et al., 1995; Masson et al., 1993; Shaywitz & Greenberg, 1999; Sheng et al., 1991) and several signalling pathways are responsible for their phosphorylation (Gupta et al., 1995; van Dam et al., 1995). These intracellular pathways are generally activated by inflammatory cytokines and cellular stress (Tibbles & Woodgett, 1999). The stress responses are adaptive processes that include apoptosis, transformation, development, immune activation, inflammation and adaptation to environmental change (Tibbles & Woodgett, 1999). A possible consequence of the decreased affinity of the CREB1–ATF1 factors for the LMP1 CRE site may be that activation of the signalling pathways in response to the environmental stress signals would not lead to an upregulation of the expression of LMP1. The ability of the latently infected B cells to maintain a low level of LMP1 despite extracellular signalling in some cases might be of advantage in EBV biology.
P3HR1 CRE-sequence variant reduces LMP1 promoter activity in a B95-8 LRS context
In a previous study of B95-8 LRS, we found that a 2 bp transverse mutation at positions –40 and –41 in the CRE site reduced EBNA2-dependent activation fivefold in the context of the wild-type +40/–106 part of B95-8 LRS (Sjoblom et al., 1998). It has also been demonstrated that a 2 bp substitution at positions –42 and –43 in the corresponding LRS CRE site of the C33A virus leads to a threefold decrease of EBNA2-induced promoter activation (Chen et al., 1995). To characterize further the consequences of sequence variation of this CRE site on LMP1 promoter transactivation, LRS from the B95-8 and P3HR1 strains cloned in luciferase reporter plasmids were co-transfected with an EBNA2 expression vector or a control vector into DG75 cells. Similar levels of EBNA2 expression in transfected cells were confirmed using immunoblot analysis (Fig. 3a). The P3HR1-derived reporter plasmid displayed approximately 50 % less activity in the presence and absence of EBNA2 in comparison to the B95-8-derived reporter plasmid (Fig. 3a). A reporter plasmid containing a B95-8 LRS region, in which the CRE element had been exchanged for the P3HR1-derived sequence [LRS B95-8 (CREP3HR1)], was induced by EBNA2 to approximately the same extent as the reporter plasmid with the P3HR1 LRS region and also had the same reduction in activity in the absence of EBNA2 (Fig. 3a). This suggests that the reduced EBNA2 response of the P3HR1 LRS reporter plasmid was due to the single base pair substitution in the CRE site. Thus, this sequence variation in the LMP1 promoter in EREB2.5 cell line should be taken into consideration in studies of the regulation of LMP1 expression employing this cell line. Overall, the results suggest that reduced ATF1–CREB1 affinity for the CRE site in LRS leads to reduced promoter activity. Given the fact that complex III had higher-binding efficiency to the P3HR1 probe (Fig. 2c), it appears from reporter experiments (Fig. 3a) as though this complex contributes to a lesser extent to EBNA2 inducibility of the LMP1 promoter and that this function is carried out mainly by the ATF1–CREB1 heterodimers. Therefore, it is likely that sequence variations in the LRS CRE site of Raji, Rael and NPC viral strains and other strains with the same substitutions will lead to decreased promoter activity.
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Taken together our results suggest that the P3HR1 CRE sequence causes a change of its factor-binding properties as compared with the corresponding B95-8 site, and as a consequence a modulation of its efficiency in activation of the LMP1 promoter. In our model system this is manifested as a reduced affinity for the ATF1–CREB1 factor complexes and a reduction of LMP1 promoter activation in the presence and absence of EBNA2. In our previous study, we showed that ATF1–CREB1 transcription factors could activate the LMP1 promoter independently of EBNA2 (Sjoblom et al., 1998). Thus, the sequence variation in this site also leads to a modulation of LMP1 expression in the absence of EBNA2.
The fact that LMP1 regulation is dependent on the cell type and latency programme is well documented (reviewed by Kieff & Rickinson, 2001). Lam et al. (2004) have reported that the levels of LMP1 vary as much as 100-fold in individual cells from the same clone and showed that the difference was because of different levels of transcript. Our study of LMP1 promoter activity in different cell lines is consistent with the notion that the activation of the LMP1 promoter is modulated both by the individual cellular context and the sequence variations and, hypothetically, epigenetic changes in the promoter. Thus, it is difficult to predict the precise physiological role of CRE-site sequence variations at different stages of EBV biology. Nonetheless, the selection of specific sequence variations in this site indicates an important regulatory function for this element in LMP1 expression. This is conceivable since a high level of LMP1 expression has been shown to induce cytostasis (Floettmann et al., 1996; Sandberg et al., 2000; Kaykas & Sugden, 2000). A high level of expression also inhibits the activity of viral and cellular promoters in the absence of cytostasis (Narbonnet & Mariame, 2006). Selection of regulatory sites with a reduced responsiveness to transcription factors without shutting it down, may regulate LMP1 expression to a steady-state level where cytostasis is prevented and cell survival is promoted. This hypothesis has been proposed by Chen et al. (1995) and is strengthened by our findings.
It is finally interesting to note that sequence variations in the LMP1-encoding region are common among EBV strains and occur independently of EBV type or origin (Jenkins & Farrell, 1996). A 30 bp deletion in the LMP1 coding sequence is so far the only sequence variation that has been functionally linked to enhanced transforming capacity (Hu et al., 1993). Selection-driven evolution of LMP1 in virus isolates from south-east Asia towards a more malignant phenotype has been suggested from phylogenetic studies (Burrows et al., 2004). Sandvej et al. (2000) have shown that the B95-8 CRE-site variant is significantly more frequent in HD virus isolates than in isolates from IM patients and asymptomatic carriers. They hypothesize that a sequence variation leading to lower LMP1 expression levels is advantageous by reducing the frequency of LMP1 driven malignancies. The question whether particular CRE-site variants in the LMP1 promoter are linked to disease remains to be answered.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 December 2006;
accepted 12 February 2007.
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