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1 Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18, Nishi-9, Kita-ku, Sapporo 060-0818, Japan
2 Department of Microbiology and Immunology, Faculty of Agriculture, Kobe University, 1-1, Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
Correspondence
Kazuhiko Ohashi
okazu{at}vetmed.hokudai.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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meq, encodes an N-terminal 98 aa of the Meq protein and lacks part of the basic leucine zipper (bZIP) and transactivation domains. In MD cell lines, transcription of L-meq was significantly downregulated, while that of the meq transcript was upregulated during apoptosis. These observations were also confirmed at the protein expression level. Reporter assays using meq- and interleukin-2 (IL-2)-promoter-driven luciferase vectors revealed that Meq suppressed transactivation by L-Meq or Meq in a dose-dependent manner. Immunoprecipitation confirmed that Meq was associated with L-Meq or Meq physically. These results suggest that Meq could be involved in apoptosis in MD cell lines as it works as a negative regulator of L-Meq and Meq by direct interaction.
The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AB091107 (L-meq) and AB091108 (meq).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). MDV strains are classified into three serotypes and only the strains of serotype 1 (MDV1) causes lymphomas (Schat, 1987). Marek's disease (MD) has been controlled by vaccination with attenuated or avirulent MDV1 and/or other serotype strains since the 1960s, and successful vaccination has been of economic benefit to the poultry industry. However, the virulence of MDV strains isolated from the field has increased and the efficacy of vaccines has decreased in the past decade due to the emergence of very virulent (vv) and very virulent plus MDV strains (Witter et al., 2005). Thus, studies for understanding the mechanisms of lymphoma formation by MDV and developing a novel vaccine strategy are required. Since MD represents the first malignant tumour controlled by vaccination, elucidating the mechanisms of tumorigenicity and the principles underlying successful vaccination may provide insights into the development of vaccines against other tumorigenic viruses such as Epstein–Barr virus (Epstein, 2001). It has been suggested that several candidate oncogenes are present in internal/terminal repeat long (IRL/TRL) regions of the MDV genome (Kung et al., 1995). Among these genes, meq, which encodes a 339 aa Meq protein with a basic leucine zipper (bZIP) motif that resembles the Fos/Jun family of oncoproteins, is consistently detected in MDV1-infected cells and tumour cell lines (Jones et al., 1992). Meq contains a proline-rich repeat (PRR) region in its C terminus and may function as both a transcriptional transactivator and a transrepressor with its resemblance to the tumour suppressor factor, WT-1 (Qian et al., 1995; Wang et al., 1993). Meq can dimerize with various factors such as c-Fos, c-Jun, ICP4 and p53 as well as Meq itself (Brunovskis et al., 1996). It was also suggested that Meq/Jun heterodimers bind to the AP-1-like sequence and activate the expression of Meq in vitro (Qian et al., 1995, 1996). The meq gene is only present in MDV1, and Meq was shown to be involved in the maintenance of the transformation status of MD tumour cell lines (Jones et al., 1992; Peng et al., 1995; Tillotson et al., 1988). Overexpression of Meq in the rodent cell line induces morphological transformation of the cells and protects the cells from apoptosis induced by tumour necrosis factor alpha (TNF-
), C2-ceramide, UV-irradiation or serum deprivation (Liu et al., 1998).
Previously, it was reported that a 180 bp sequence is inserted into the region encoding the C-terminal transactivation domain in the meq open reading frame (ORF) of an attenuated vaccine strain, CVI988 (Lee et al., 2000). This gene, termed L-meq, was also detected in chickens infected with vvMDV1 strains, Md5 and RB1B, during latency (Chang et al., 2002a). The inserted 180 bp sequence encodes three additional copies of PRR and, as a result, L-Meq suppressesed transactivation by Meq and may partially contribute to the protection of MD by vaccination (Chang et al., 2002b). The meq gene has been known to be polymorphic and several variants have been reported, including L-meq (Chang et al., 2002c). The presence of multiple spliced transcripts of meq has also been suggested in several studies (Jones et al., 1992; Peng & Shirazi, 1996; Le Rouzic et al., 2002). Nevertheless, the functions of Meq and its variants in the establishment of latency or transformation have not been elucidated clearly.
In this study, we identified a novel spliced form of the meq transcript, termed meq, as well as the L-meq transcript in MD cell lines. We speculate that Meq could work as a negative regulator of Meq and L-Meq, and that Meq could be involved in the loss of the transformed phenotype of MD cell lines because the putative Meq protein lacks a part of bZIP and transactivation domains. Thus, we analysed the expression of the meq transcripts and the Meq proteins in MD cell lines during apoptosis, and investigated the involvement of these meq variants in maintenance of the transformed phenotype. As shown herein, the expression of the Meq protein was upregulated during apoptosis, and Meq would in part repress transactivation by L-Meq or Meq by physical association, suggesting that Meq could play a role in apoptosis in MD cell lines.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), MSB1-cl.18, a transplantable subclone derived from MSB1-O (Higashihara et al., 1984), MTB1-cl.21 (Ikuta et al., 1987) and HP1 (Powell et al., 1974), were maintained at 41 °C, 5 % CO2 in RPMI 1640 (Sigma) supplemented with 10 % fetal calf serum (FCS; Gibco). Chick embryo fibroblasts (CEF) were prepared from 11-day-old fertile eggs (Shiroyama Farm of Breeding Hens Inc.) and maintained at 37 °C in Eagle's MEM (Nissui) supplemented with 10 % tryptose phosphate broth (Difco Laboratories) and 10 % FCS. The immortalized CEF cell line, DF-1 (Himly et al., 1998), was maintained at 39 °C in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10 % FCS.
Virus.
A strain of vvMDV1, Md5 (Witter, 1983), was obtained from chicken kidney cell culture of experimentally infected chickens in our laboratory. This virus was propagated in the CEF monolayer. The virus was inoculated onto CEF cells at 100 p. f. u. per well in a six-well plate format and infected cells were harvested 24 h after virus inoculation.
Cellular DNA extraction.
MD cell lines, CEF (about 5x106 cells) infected with Md5 (CEF/Md5) and uninfected CEF were suspended in lysis buffer (20 mM Tris pH 8.0, 25 mM EDTA pH 8.0, 100 mM NaCl and 0.5 % SDS) containing 50 mg proteinase K ml–1, and incubated at room temperature with gentle rotation for an overnight period. After treatment with RNase, total cellular DNA was extracted with phenol/chloroform-isoamylalcohol (25 : 24 : 1) and precipitated with ethanol. The resultant DNA was dissolved in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0).
Induction of apoptosis in MD cell lines.
Approximately 3x106 cells per 5 ml medium of MD cell lines were treated with either actinomycin D (2 mg ml–1), etoposide (50 µM), cycloheximide (100 µg ml–1) or dexamethasone (100 µM). Cells were then harvested after 3, 6 and 9 h (actinomycin D, etoposide) or 6, 12 and 24 h (cycloheximide, dexamethasone). MD cell lines were also cultured under serum-starved conditions (1 or 0.5 % FCS) and harvested after 48 h. Cells were washed twice with PBS (pH 7.2) and used for the extraction of total cellular RNA, or lysed in 2x SDS sample buffer for Western blot analysis as described below. Apoptosis was confirmed by the detection of DNA fragmentation as described previously (Takagi et al., 2006).
Extraction of total cellular RNA and cDNA synthesis.
MD cell lines untreated or treated with either cycloheximide or dexamethasone were resuspended in 1 ml of the TRIzol reagent (Invitrogen), and total cellular RNA was extracted as indicated by the manufacturer. Total cellular RNA of CEF/Md5 and uninfected CEF was extracted as well. Each RNA sample was treated with 10 U DNase I (Promega) and was incubated at 70 °C for 15 min for denaturation. Reverse transcription was carried out in a final volume of 30 µl of reaction buffer containing 50 mM Tris/HCl (pH 8.3), 40 mM KCl, 1 mM DTT, 3 mM MgCl2, 0.5 mM each of dNTPs, 40 U RNase inhibitor (Promega), 200 pmol oligo(dT)15 and 20 U of the RAV-2 reverse transcriptase (Takara). The mixture was incubated at 42 °C for 60 min for reverse transcription, then at 70 °C for 10 min. Synthesized first strand cDNA (30 µl) was diluted with distilled water up to 100 µl.
Semi-quantitative RT-PCR.
Synthesized cDNAs were used to amplify the meq-specific sequences. To standardize the quantity of each cDNA, the expression of the 
-actin transcript was measured as an internal control. The primers used for meq-specific PCR were M-S (5'-ATGTCTCAGGAGCCAGAGCCGGGCGCT-3') and M-AS (5'-GGGGCATAGACGATGTGCTGCTGAG-3') as described previously (Lee et al., 2000). We also employed primers MR-S (5'-AGTTGGCTTGTCATGAGCCAG-3') and MR-AS (5'-TGTTCGGGATCCTCGGTAAGA-3'), which amplify the C terminus coding region of meq (Chang et al., 2002a) for specific quantification of the L-meq transcript. For the -actin amplification, sense (5'-ACGTCGCACTGGATTTCGAG-3') and antisense (5'-TGTCAGCAATGCCAGGGTAC-3') primers were used. PCR was performed in a final volume of 20 µl containing 1.5 mM MgCl2, 0.25 mM of each primers, 0.1–0.5 µg of cellular DNA or cDNA samples, 0.2 mM of dNTPs and 0.25 U Taq polymerase (Takara). PCR reaction was carried out as follows: 1 cycle of a denaturation step at 95 °C for 5 min, 20 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for 30 s (for -actin) or 30 cycles of denaturation at 95 °C for 40 s, annealing at 56 °C for 40 s and extension at 72 °C for 1 min 20 s (for meq) followed by the final extension step at 72 °C for 7 min. PCR products (10 µl) were separated on a 1.5 (-actin) or 1.2 % (meq) agarose gels and visualized by ethidium bromide staining.
Sequencing of the L-meq and 0.4 kb PCR product.
The PCR products corresponding to the L-meq and 0.4 kb fragment (termed meq) were extracted from the gel and purified using the Geneclean II kit (Bio 101) and cloned into the pGEM-T easy vector (Promega). The constructed plasmids (designated pGEM-Te-L-meq and pGEM-Te-meq) were purified by a standard mini-prep method followed by polyethylene glycol precipitation and sequenced by using the Thermo Sequenase Core Sequencing kit (Amersham Life Sciences) and DNA sequencer SQ-5500 (Hitachi).
Bacterial expression of Meq fusion proteins and generation of rat antisera.
The meq gene of the Md5 strain was amplified by PCR using specific primers, gel-purified and cloned into the pGEM-T easy vector. The resultant plasmids designated pGEM-Te-Md5meq and pGEM-Te-meq were digested with EcoRI, and subfragments containing Md5meq or meq were cloned into the EcoRI site of the pET TRX Fusion System 32b (Novagen). Fusion proteins were expressed in a bacterial host, AD494 (DE3) pLysS, by induction with 1 mM isopropyl -D-thiogalactopyranoside for 9 h. Proteins were purified using the His-Bind Resin and Buffer kit (Novagen) as instructed by the manufacturer. Female Wister rats (12 weeks old) were injected intramuscularly with 100 µg of the entire fusion proteins with complete Freund's adjuvant, and boosted at days 14 and 21 after the first immunization followed by intravenous immunization on day 28. Antisera were collected 14 days after the final boost.
Western blot analysis.
Western blot analysis was performed as follows: cell lysates (approx. 1x106 cells per 10 µl) were subjected to SDS-PAGE (13 % polyacrylamide) and transferred onto the Immobilon Transfer membranes (Millipore). The blots were incubated with primary antibody (rat antisera at a 1 : 1000 dilution) for 1 h, washed three times with PBS containing 0.05 % Tween 20 (PBS-T). Then, the blots were incubated with peroxidase-conjugated goat anti-rat IgG (Cosmo Bio) for 30 min and washed three times with PBS-T. Finally, the blot was incubated with 0.5 mg 3,3-diaminobenzidine tetrahydrochloride ml–1 in PBS solution supplemented with 1/1000 volume of 30 % hydrogen peroxide for detection. The blot was also probed with mouse anti-actin monoclonal antibody (Chemicon International) followed by peroxidase-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch) to verify the amount of proteins loaded and transferred.
Construction of the expression plasmids.
The L-meq, meq and meq transcripts were amplified by PCR using primers M-S-XhoI, which contains the XhoI site at the 5' end, and M-AS. Chicken c-Jun transcript was also amplified by PCR using primers cJ-S-XhoI (5'-CTCGAGAAGATGGAGCCTACTTTCTACGA-3') and cJ-AS (5'-GTTTGGTTATACCACAACATCACAG-3'). The amplified fragments were gel-purified and cloned into the pGEM-T easy vector, and digested with restriction enzymes XhoI and NotI. The XhoI–NotI fragments were cloned into the XhoI and NotI sites of the eukaryotic expression vector pCI-neo (Promega), and expression plasmids designated pCI-L-Meq, pCI-Meq, pCI-Meq and pCI-c-Jun were constructed. The meq transcripts were also amplified using primers M-S-BglII and M-AS-XhoI, gel purified and digested with BglII and XhoI. These fragments were cloned into the BglII and XhoI sites of the pCMV-Tag1 vector (Stratagene) to construct the expression plasmids of N-terminal FLAG-tagged Meq proteins. These plasmids were designated pCMV-L-Meq, pCMV-Meq and pCMV-Meq, respectively.
Dual luciferase reporter assay.
The promoter region of the meq gene was amplified as described by Levy et al. (2003), and that of the IL-2 gene (–578/+34) was also amplified by PCR. These fragments were cloned upstream of the firefly luciferase gene in the pGL3-Basic vector (Promega). The resultant plasmids, designated pGL3-MP and pGL3-IL2P, were used as reporter plasmids. DF-1 cells were seeded in 24-well plates at 3x105 cells in 0.5 ml DMEM containing 10 % FCS and incubated overnight at 39 °C. Transfection was carried out using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction. For each well, a total amount of 0.8 µg of the vectors including pGL3-MP or pGL3-IL2P expression vectors and pRL-tk, which served as an internal control reporter, were transfected. After 24 h incubation, cells were lysed with 1x Passive Lysis buffer, and a dual luciferase assay was performed according to the manufacturer's instructions using Luminescencer-JNR AB-2100 (Atto). The luminescence intensity of firefly luciferase was normalized to that of the Renilla luciferase.
Co-immunoprecipitation.
Co-immunoprecipitation of L-Meq/Meq with Meq was analysed as follows. Approximately 80 % confluent DF-1 cells in 12-well plates were transfected with 0.8 µg of each pCMV-Tag1 or pCMV-Meq in combination with pCI-L-Meq or pCI-Meq using the Lipofectamine 2000 reagent. After 24 h incubation, cells were lysed with high salt RIPA buffer (50 mM Tris pH 8.0, 500 mM NaCl, 1 % NP-40, 0.25 % sodium deoxycholate and 1 mM EDTA pH 8.0) supplemented with Complete Protease Inhibitor Cocktail (Roche). Insoluble debris was removed by centrifugation, and the supernatants were pre-cleared using Protein A/G Plus Agarose (Santa Cruz) combined with mouse naïve serum by gentle rotation for 1 h at 4 °C. After centrifugation, supernatants were transferred into new tubes. FLAG-tagged Meq and interacting proteins were co-precipitated using anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) conjugated with Protein A/G Plus Agarose. Immunoprecipitates were washed six times with high salt RIPA buffer and eluted in 30 µl of 2x SDS sample buffer. After boiling, eluates were subjected to SDS-PAGE (12.5 % polyacrylamide), and Western blot analysis was performed using anti-Meq serum as described above.
Co-immunoprecipitation of c-Jun with Meq or its variants was analysed by the same method, using pCMV expression vectors and pCI-c-Jun. The blot was probed with rabbit anti-c-Jun polyclonal IgG (Santa Cruz) followed by peroxidase-conjugated goat anti-rabbit IgG (Cappel).
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RESULTS |
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). The meq-specific PCR was also performed using cDNA samples synthesized from total cellular RNA of MD cell lines and CEF/Md5 to detect the meq transcripts. L-meq transcript and an approximately 0.4 kb fragment were detected in MD cell lines, while meq transcript and a 0.4 kb fragment were detected in CEF/Md5 (Fig. 1b).
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meq, was a newly identified form of the meq transcript that encodes part of the Meq protein. The meq encodes an N-terminal region of 98 aa of Meq and a frame-shifted different C terminus of 30 aa. Putative Meq protein lacks part of the bZIP and transactivation domains (Figs 2 and 3). The meq transcript detected in CEF/Md5 has almost the same sequence as that of MSB1-O (data not shown), suggesting that this short form is an alternatively spliced product of both L-meq and meq.
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). Cells were harvested and the expression of the meq transcripts during the treatments with cycloheximide and dexamethasone were evaluated by semi-quantitative RT-PCR. The L-meq and meq transcripts were consistently present in untreated MD cell lines (Fig. 4b). In both of the cell lines treated with cycloheximide, the expression of the L-meq transcript was downregulated and finally under the detectable level during apoptosis. This result was also confirmed by specific amplification of 0.8 kbp fragment of L-meq using MR primers described in Methods. In contrast, the meq expression was remarkably upregulated during apoptosis (Fig. 4b, indicated as ‘CHX’). Apoptosis was not induced with dexamethasone, and the expression of the L-meq and meq transcripts was not altered after drug treatment in MD cell lines (Fig. 4b, ‘Dex’).
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Meq (rMeq) proteins. The putative 70 kDa L-Meq protein was successfully detected in MD cell lines with both of the antisera, while no specific band was detected with pre-immune serum (Fig. 5a, upper panels). In addition, an approximately 18 kDa band corresponding to the Meq protein was detected with anti-rMeq antiserum (Fig. 5a, lower panels). Therefore, in the following experiment, we used anti-rMeq antiserum for the detection of the L-Meq protein and anti-rMeq antiserum for the detection of the Meq protein.
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, indicated as ‘Cont.’). In MSB1-O, a decrease in the L-Meq expression and an increase in the Meq expression were observed after the induction of apoptosis with either actinomycin D, etoposide or cycloheximide (Fig. 5b). Similarly, the L-Meq expression was decreased and Meq expression was increased in HP1 during apoptosis induced with either actinomycin D or cycloheximide. In this experiment, as mentioned above, etoposide did not induce apoptosis in MD cell lines effectively, but a slight increase of the Meq expression was observed in etoposide-treated cells (Fig. 5b). Dexamethasone did not induce apoptosis in MD cell lines, and no significant change was observed in the expression of the Meq proteins (Fig. 5b). No notable change was observed in the L-Meq expression, but an increase in the Meq expression was observed in cell lines cultured under serum-starved conditions (Fig. 5b, 1 and 0.5 % FCS).
The effect of Meq on transactivation by Meq and L-Meq
In our previous study, transactivation by L-Meq and Meq on the meq promoter has been tested by using a luciferase assay (Chang et al., 2002b). L-Meq itself functioned as a transactivator, but its transactivation was lower than that of Meq, suggesting a suppressive effect of L-Meq on Meq transactivation. It has been reported that Meq, as a heterodimer with c-Jun, could recruit the chicken IL-2 promoter, which has an AP-1 site (Levy et al., 2003). In this work, the effect of Meq on transactivation by L-Meq or Meq was examined by using meq or IL-2-promoter-driven luciferase reporter vectors. As shown in Fig. 6(a), both L-Meq and Meq enhanced transactivation on both of the promoters, and transactivation by L-Meq was lower than that by Meq, comparable to our previous study. In contrast, Meq did not transactivate these promoters, as expected from its structure. Co-transfection experiments showed that Meq negatively regulated the transactivation by L-Meq and Meq in a dose-dependent manner (Fig. 6b).
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Meq to L-Meq/Meq and c-JunMeq, we examined whether Meq can interact with L-Meq, Meq and the cellular bZIP protein c-Jun by co-immunoprecipitation. The FLAG-tagged Meq plasmid (pCMV-Meq) or control plasmid (expressing only the FLAG tag peptide) was transfected in combination with native L-Meq or Meq plasmid (pCI-L-Meq or pCI-Meq) into DF-1 cells. L-Meq, Meq and FLAG–Meq were successfully expressed in transfected cells, as confirmed by Western blot analysis (Fig. 7a, upper panels). The result of co-immunoprecipitation revealed that L-Meq as well as Meq were co-precipitated along with FLAG–Meq. By contrast, neither L-Meq nor Meq was precipitated in the absence of FLAG–Meq (Fig. 7a, lower panels). Next, the FLAG-tagged Meq and its variants were expressed in combination with c-Jun. We overexpressed c-Jun by co-transfection of the pCI-c-Jun plasmid because endogenous expression of c-Jun was moderate in DF-1 cells (Fig. 7b ‘Cont.’). As shown in Fig. 7(b), c-Jun was co-precipitated along with Meq, comparable to the results described by Qian et al. (1995). L-Meq was also able to interact with c-Jun, as expected from its structure containing the entire bZIP domain. On the other hand, c-Jun was not co-precipitated along with Meq, showing that Meq did not interact with c-Jun.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Its gene product, Meq, is a transcription factor with an N-terminal bZIP domain homologous to Jun/Fos oncoproteins (Jones et al., 1992). Meq can interact with itself and cellular bZIP proteins such as c-Jun and c-Fos as well as p53 and C-terminal-binding protein, and can contribute to cellular immortalization (Brunovskis et al., 1996; Brown et al., 2006). A recent study also demonstrated that Meq is a transforming protein that activates transformation-related genes such as JTAP-1, JAC and HB-EGF as well as cell survival pathways, by upregulating proto-oncogenes such as bcl-2, c-jun and c-ski (Levy et al., 2005).
It has been noted that meq is a polymorphic gene. Our previous study reported the presence of the L-meq gene in an attenuated vaccine strain, CVI988 (Lee et al., 2000). The L-meq gene has also been identified in some other mild/virulent MDV1 strains (Shamblin et al., 2004). Interestingly, the L-meq gene was also detected in chickens infected with very virulent MDV1 at 3–5 weeks post-infection (Chang et al., 2002a). We have also shown the presence of other forms of the meq gene, S-meq and VS-meq with sequences encoding different copy numbers of the PRR region (Chang et al., 2002c). The biological significance of the coexistence of meq, L-meq and other forms of meq in MDV-infected chickens has not been elucidated clearly, but L-Meq could exert an inhibitory effect on the transactivation by Meq and may suppress the replication of the oncogenic MDV1 (Chang et al., 2002b).
Several studies have shown the presence of multiple forms of the meq transcript (Jones et al., 1992; Le Rouzic et al., 2002). One of these transcripts, termed Meq-sp, was initially identified in the MD cell line, MKT-1 (Peng et al., 1995). Meq-sp is encoded from an alternatively spliced transcript, and its C terminus is replaced by peptides encoded by the second and the third exons of vIL-8 (Liu et al., 1999). A recent study reported two other spliced variants of meq. One of them was termed Meq/RLORF5a, which encodes the N terminus of Meq and a part of RLORF5a, and the other had an uncharacterized sequence downstream of the meq gene (Jarosinski & Schat, 2007). In this study, we have identified another spliced transcript, meq, and examined the expression of the meq transcripts and Meq proteins during apoptosis to investigate the function of L-Meq and Meq in MDV-transformed cell lines.
The meq transcript consists of nt 1–294 and 915–1020 of meq ORF, and encodes an N-terminal 98 aa of Meq and a frame-shifted C-terminal 30 aa (Figs 2, 3 and refer to GenBank accession no. AB091108). meq could be an alternatively spliced transcript because the assumed intron (nt 295–914) has possible CT–AC donor and acceptor sites, which are non-canonical but have been reported in the HHV-6 U83 gene (French et al., 1999). However, the meq transcript was not detected in DF-1 cells transfected with pCI-Meq or pCI-L-Meq (data not shown), indicating that some other viral factor(s) could be involved in the splicing of the C-terminal coding region of meq or L-meq. In MD cell lines, L-meq and meq transcripts were detected. Western blot analysis confirmed that the L-meq transcript was expressed as a putative L-Meq protein, and that its expression was decreased during drug-induced apoptosis. The meq transcript could also be translated to generate an 18 kDa Meq protein as predicted from its sequence. The Meq expression was remarkably increased during drug-induced apoptosis as well as serum-starvation, despite the defective induction of apoptosis (Fig. 5).
It has been reported that Meq homodimer binds to the ACACA motif of the MDV origin of replication (MDV-Ori), and represses the flanking bi-directional promoters overlapping MDV-Ori and regulates the replication of MDV (Levy et al., 2003; Parcells et al., 2003). It was also suggested that Meq-sp was dominantly expressed during the lytic replication phase of MDV, as a putative negative regulator of the functions of Meq (Peng & Shirazi, 1996; Parcells et al., 2003). A recent study described the distinct mobility and the different functions between Meq and Meq-sp (Anobile et al., 2006). The Meq also lacks the transactivation domain and predominates during apoptosis in MD cell lines. Therefore, we have speculated that Meq may exert a negative effect on L-Meq or Meq functions such as transactivation. As expected, Meq inhibited the transactivation by L-Meq or Meq through the meq and IL-2 promoters in a dose-dependent manner, while its inhibitory effect may be marginal. The third leucine in the bZIP domain of Meq is replaced by an arginine, a highly hydrophilic residue (Fig. 2). Hence, Meq would be able to form only one loop of helical conformation that seems insufficient to heterodimerize via the bZIP domain. However, our results showed that Meq interacts with L-Meq and Meq, but not with c-Jun (Fig. 7). The mechanisms underlying this ‘selective interaction’ is unknown, and we could not detail how Meq could be associated with L-Meq and Meq. Nevertheless, Meq could exert its inhibitory effect by direct interaction with L-Meq and Meq. Further studies such as chromatin-immunoprecipitation or electrophoretic mobility shift assay are required to investigate detailed mechanisms of interaction and precise roles of Meq. L-Meq may be involved in the MDV latency as suggested previously (Chang et al., 2002a). In addition, L-Meq may also play a role in cellular transformation, as Meq does, because it exhibited transactivation of both of the promoters used in this study, although its activity was lower than that of Meq. Thus, the downregulation of L-Meq as well as the upregulation of Meq could be involved in cellular apoptosis. Meq may also play a role in the lytic infection, part of the reason for this speculation is that it predominates in vvMDV-infected CEFs when the cytopathic effect was confluent (data not shown).
In summary, we have reported a novel spliced transcript termed meq. Our results demonstrated that Meq would be a negative regulator of the functions of L-Meq and Meq, and may be involved in apoptosis in MD cell lines. Further study for details of expression manners, interaction partners and functions of Meq variants both in vitro and in vivo will contribute to understanding the mechanisms of MDV oncogenicity and transformation.
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Received 29 November 2006;
accepted 17 April 2007.
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P<0.05 by unpaired t-test.