Experimental Cell Research 303 (2005) 47 – 55 www.elsevier.com/locate/yexcr
Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein Elena Kashubaa,*, Mariya Yurchenkob, Krisztina Szirakc, Joachim Stahld, George Kleina, Laszlo Szekelya a Microbiology and Tumor Biology Center (MTC), S-171 77 Stockholm, Sweden Institute of Experimental Pathology, Oncology and Radiobiology (IEPOR) NAS of Ukraine, 03022 Kiev, Ukraine c University of Debrecen, Medical and Health Science Center, Department of Human Genetics, H-4012 Debrecen, Hungary d Max Delbruck Center of Molecular Medicine, D-13125 Berlin, Germany b
Received 2 April 2004, revised version received 13 July 2004 Available online 18 September 2004
Abstract Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fosmediated cellular transformation and is part of the small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a. D 2004 Elsevier Inc. All rights reserved. Keywords: Epstein-Barr virus; EBNA-5; Fte-1/S3a ribosomal protein; Cell transformation; Yeast two-hybrid system
Introduction Epstein-Barr virus (EBV) is a lymphotropic gammaherpes virus that infects more than 90% of the human population and targets B cells for infection. EBV causes B cell lymphomas in immunosuppressed hosts, such as transplant recipients (posttransplant lymphoproliferative disease, PTLD) and AIDS patients. EBV is associated with several malignancies, particularly with Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC). * Corresponding author. Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Nobels vag 16 Box 280, S-171 77 Stockholm, Sweden. Fax: +46 8 330498. E-mail address:
[email protected] (E. Kashuba). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.08.025
EBV infection transforms B cells into large immunoblasts that express six EBV-encoded nuclear antigens (EBNA-1-6), three membrane proteins (LMP-1, -2A, -2B) and two small non-polyadenylated RNAs (EBER-1-2). Five of them—EBNA-2, -3, -5, and -6 and LMP-1—are required for immortalization of B cells (for review, see Ref. [1]). EBNA-5 (also called EBNA-LP) is, together with EBNA-2, the earliest viral protein expressed in freshly infected B cells [2,3]. These two proteins together can induce G0 to G1 transition in resting B cells [4]. It was shown that coexpression of EBNA-5 with EBNA-2 increases the transactivating power of EBNA-2 [5,6]. EBNA-5 is a nuclear phosphoprotein that is tightly associated with the nuclear matrix [7,8]. Several interaction partners of EBNA-5 have already been identified. The
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soluble, detergent-eluable fraction of EBNA-5 was found to be coimmunoprecipitated with the heat shock protein Hsp70 [9,10]. Flag-labeled EBNA-5 could precipitate HA95 (AKAP95, A-kinase anchoring protein 95), small ATPindependent chaperone Hsp27, prolyl 4-hydroxylase alpha subunit, alpha-tubulin, and beta-tubulin from the lymphoblastoid cell lysates [11]. Yeast two-hybrid screening identified the cytoplasmic Hax-1 protein [12,13] and HERR-1 (human estrogen-related receptor 1) [14] as binding partners. We have recently found that EBNA-5 also binds to the nucleolar p14ARF protein, an upstream regulator of the p53 pathway [15]. Co-transfection of p14ARF and EBNA-5 led to accumulation of these proteins in the nuclear precipitates, where they were colocalized. EBNA-5 prolonged the survival of p14ARF-transfected cells. We now report the identification of another human protein with partially nucleolar localization, Fte-1/S3a, as an EBNA-5 binding partner. This protein was first identified as v-fos transformation effector (Fte-1) [16]. Later, it was shown that Fte-1 is identical to the protein S3a, an important component of the small ribosome subunit. S3a is directly involved in eukaryotic protein synthesis by interacting with 3Vregion of 18S rRNA, mRNA, initiation factors eIF-2 and -3, and elongation factors EF-1 and -2 (for review, see Ref. [17]). In this way, Fte-1/S3a plays a central role in the regulation of translation. In addition, the overexpression of Fte-1/S3a is associated with a rapid cell proliferation, maintenance of undifferentiated state, and proneness for apoptosis [18].
Yeast strains and cDNA library screening The Saccharomyces cerevisiae HF7c strain was used for library screening. SFY526 strain was used to confirm the interaction. Human lymphocyte MATCHMAKER cDNA library in pACT GAL-4 transcriptional activation domain vector and the yeast strains were obtained from Clontech. Library screening was run according the Clontech protocol. Interacting clones were selected on SD plates lacking His, Leu, and Trp. The fastest growing clones were further tested for h-galactosidase activity by ONPG test as described [22]. Specific activity of the given clones was calculated as percentage of h-galactosidase units of the positive control. The samples were incubated with ONPG at 308C for 2 h. Sequencing Sequencing was done using capillary Apply BioSystem sequence machine (Perkin-Elmer). PCR PCR was carried using Idahotech thermocycler. 5Vprimer for Fte-1/S3a cloning, sequence (5Vto 3V): TAATTGGATCC AT G G C G G T T G G C A A G A A C A A G C G C C T TA C GAAAGGCGGCAAAAA 3Vprimer for Fte-1/S3a cloning, sequence (5V to 3V): CGTGAATCCGCCACTATTTGGAGTCTGAACTTTA. Primers were obtained from GIBCO BRL. First cycle was the following: 948C for 4V, 658C for 20V, and 728C for 2V; 35 cycles of 948C for 10U, 658C for 20U, and 728C for 40U were run, ending with 728C for 7V.
Materials and methods Cells and cell culture Plasmids Construction of the plasmids in GAL4-binding domain containing vector, BD (Clontech) BD-EBNA-1, BD-EBNA3, BD-DEBNA-4, BD-EBNA-5 was described previously [19]. Cloning of GST2TK-EBNA-5, CMV-EBNA-5, and pBabe-puro-EBNA-5 was discussed in [20]. Construction of GFP-EBNA-5 was mentioned in Ref. [21]. All EBNA-5 constructs contained four W1W2 repeats and the unique Cterminal domain (Y). GST2TK-EBNA-5 mutants that contained the Y domain and a varying number of W1W2 repeats (1, 2, and 4), as well as a construct that lacked the Y but contained the four W1W2 repeats, were described in [20]. Mouse p53, lacking N-terminus, in pGBT9 (pVA3) and SV40 Large T-antigen in pGAD10 (pTD1, both from Clontech) were used as positive interaction controls. The GFP-Fte-1/S3a was generated by inserting of the 835 bp long PCR product, digested in BamH1/EcoRI sites into GFP vector, cleaved with BglI/EcoRI. GST2TK-Fte-1/S3a was created similarly, except that GST2TK was digested in BamH1/EcoRI site. Primers for cloning and cloning strategy are described below.
MCF7 breast carcinoma cell line was cultured at 378C, in Iscove medium containing 10% fetal bovine serum. Periodic staining with Hoechst 33258 (bisbenzimide) monitored the absence of mycoplasma. The cells were grown on the cover glass. We transfected cells with GFP-Fte-1/S3a, pBabeEBNA-5, GFP-EBNA-5, and CMV-EBNA-5 constructs using Lipofectamine Plus Reagent (Life Technology) according to the manufacturer’s protocol. Tonsil B cells were isolated from human tonsils obtained from routine tonsillectomy (Karolinska Hospital, Stockholm). The tonsils were cut into the fragments and passed through a metal mesh. Mononuclear cells were isolated on Lymphoprep gradients. The two subsequent rounds of Erosetting removed T cells. Cells were anchored onto glass slides using Cytospin cytocentrifuge. GST pull-down assay GST pull-down assay was performed as described [23]. GST-fusion proteins on beads were treated with RNAse A and DNAse. All of the cell lysates contained 0.5% of BSA
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as nonspecific competitor. We have probed Western blots with mouse monoclonal antibody against EBNA-5 JF186 [2] and with antibody against Fte-1/S3a that was raised in goat or rabbit [24]. Immunofluorescence staining Immunostaining and digital image capturing were carried out as described [25]. We have used the anti-EBNA-5 monoclonal antibody JF186 and rabbit anti-Fte-1/S3a antibody. As the secondary antibodies, the FITC- and TRITCconjugated swine anti-rabbit (DAKO), Texas red-conjugated horse antimouse (Vector Lab), TRITC-conjugated goat antirabbit (Sigma), FITC-conjugated goat antimouse (Sigma) have been used. Hoechst 33258 was added at the concentration of 0.4 Ag/ml to the secondary antibody for DNA staining.
Results EBNA-5 binds to Fte-1/S3a in the yeast two-hybrid system Screening of a human lymphoblast cDNA library with EBNA-5/BD construct identified a clone, named S1, that could grow on His-, Leu-, and Trp-deficient SD medium. This clone expressed h-galactosidase from a lacZ reporter, activated by protein–protein interaction. The strength of the binding was about 15% of the very strong positive pRbSV40LT control. The S1 clone contained the i900-bp-long insert at its XhoI site. The interaction between EBNA-5 and S1 was confirmed by re-transformation of the corresponding plasmids into the SFY526 yeast strain. Empty pGBT9/BR vector and DEBNA-3, DEBNA-4, EBNA-1 constructs in the GAL4binding domain yeast vector were used as negative controls. None of them showed any significant interaction, according to the h-galactosidase test. Sequencing of the insert showed that the EBNA-5 interactive clone was identical to the human v-fos transformation effector Fte-1 (GenBank accession no. AAA58487). The protein is also known as the ribosomal protein S3a (GenBank accession no. P49241). 5Vof Fte-1/S3a, encoding the first 14 residues, was missing from the cDNA library insert. PCR amplification was used to obtain the full-length Fte1/S3a gene. We have designed a long (55 bp) forward primer to build up the 5Vend. The full-length cDNA, encoding a 264amino acid (aa) protein, was cloned into green fluorescence protein fusion vector pEGFP-C1 (GFP) and into glutathioneS-transferase bacterial expression vector (GST2TK). EBNA-5 binds Fte-1/S3a in vitro Bacterial-produced GST2TK-Fte-1/S3a, GST2TKEBNA-5, and GST2TK proteins were used to precipitate
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the cell lysates of a freshly established lymphoblastoid cell line (LCL). GST2TK-EBNA-5 fusion protein on the surface of the glutathione sepharose beads precipitated Fte-1/S3a protein as was shown by Western blotting (Fig. 1A). To answer the question whether the full length of EBNA-5 or the specific part of protein is needed for the interaction with Fte-1/S3a, a series of EBNA-5 deletion mutants were used. We have used the four EBNA-5 mutant constructs: (a) three of them that contained the unique C-terminal domain (Y) and a varying number of W1W2 repeats (1, 2, and 4); (b) one construct that lacked the Y but contained the four W1W2 repeats. None of the mutant EBNA-5 constructs precipitated Fte-1/S3a from the cell lysates (data not shown). This suggested that four repeats and an intact Cterminal unique region are required for the Fte-1/S3a binding. It is worth to mention that the deletion of Cterminal part of EBNA-5 results in the inhibition of EBVinduced B cell transformation. To confirm an in vitro binding between Fte-1/S3a and EBNA-5, we have used GST2TK-Fte-1/S3a fusion protein on beads to precipitate EBNA-5 from the LCL lysates. We have observed that GST2TK-Fte-1/S3a, but not GST2TK, precipitated EBNA-5 from the LCL lysates (Fig. 1B). However, the amount of EBNA-5, precipitated with GST2TK-Fte-1/S3a, was less than total amount of EBNA5 in the cell lysates. Probably, we could not release EBNA-5 completely from nuclear matrix, to which it tightly bound, using the NP40 lysis buffer [7].
Fig. 1. (A) GST pull-down assay. Western blotting shows that GST2TKEBNA-5 but not GST alone precipitates Fte-1/S3a from the LCL lysates. (B) GST pull-down assay. Western blotting shows that GST2TK-Fte-1/S3a but not GST2TK precipitates EBNA-5 from LCL lysates.
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Fig. 2. (A) pBabe-EBNA-5 and endogenous Fte-1/S3a colocalize in the nucleoli of the transfected cells. MCF7 cells were transfected with pBabe-EBNA-5 construct. Endogenous Fte-1/S3a protein (red) and EBNA-5 (green) colocalize in the nucleoli (arrows) of the transfected cells. DNA staining is shown in blue. (B) GFP-EBNA-5 and endogenous Fte-1/S3a colocalize in the nucleoli of the transfected cells. Endogenous Fte-1/S3a (red) and GFP-EBNA-5 (green) colocalize in the nucleoli (arrows) of the transfected cells. Blue—DNA staining. (C) EBNA-5 and GFP-Fte-1/S3a colocalize in extranucleolar sites in the nucleus of the transfected cells. MCF7 cells were cotransfected with GFP-Fte-1/S3a (green) and CMV-EBNA-5 (red). EBNA-5 that expressed constitutively from pBabe-EBNA-5 construct is shown on the left panels. Transiently transfected CMV-EBNA-5 is shown on the right panel. Overexpression of both Fte-1/ S3a and EBNA-5 proteins leads to the formation of double-positive nuclear inclusions (arrows). DNA staining is shown in blue.
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Fig. 2 (continued).
Endogenous Fte-1/S3a colocalizes with EBNA-5 in the nucleus of the EBNA-5-transfected cells MCF7 cells that express low levels of wild-type p53, MDM2, and p21 were transfected with two EBNA-5 constructs—pBabe-EBNA5 where EBNA-5 is transcribed from the Moloney virus LTR promoter and GFP-EBNA-5 that expresses the GFP fusion protein from a stronger CMV promoter. The endogenous Fte-1/S3a protein was stained with rabbit monospecific antibody. It was localized in both the nucleus and the cytoplasm. In the nucleus, it preferentially accumulated in the nucleoli. The EBNA-5-transfected cells showed that both the endogenous Fte-1/S3a protein and EBNA-5 have localized to the nucleoli (Figs. 2 A and B). Importantly,
EBNA-5 expression with nucleolar localization has led to the enrichment of the endogenous Fte-1/S3a in the nucleoli. GFP-Fte-1/S3a and EBNA-5 colocalize in the nucleus of the transfected cells Exogenous EBNA-5 that was expressed in MCF7 cells from either pBabe-EBNA-5 or CMV-EBNA-5 vectors showed a rather homogeneous distribution in the nucleus with occasional accumulation in the nucleoli. GFP-Fte-1/S3a protein transfected into MCF7 cells showed somewhat altered cellular distribution compared to the endogenous Fte-1/S3a protein. It was almost not present in the cytoplasm but accumulated in the nucleoli of the transfected cells. Cells that expressed high levels of GFP-
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Fte-1/S3a often contained nuclear inclusions outside the nucleoli (Fig. 2C). In spite of the lack of GFP-Fte-1/S3a in cytoplasm, we wanted to study the relationship between this protein and EBNA-5 that was also expressed from the strong promoters. In cells that were double-transfected with GFP-Fte-1/S3a and CMV-EBNA-5, the two proteins showed high degree colocalization in the nuclear (extranucleolar) inclusions (Fig. 2C). The same phenomenon was also observed in Cos-7 cells (data not shown). EBV induces Fte-1/S3a upon infection of tonsil B cells Fte-1/S3a levels are often greatly increased in different malignantly transformed cells [26]. To answer the question whether upon EBV-mediated transformation Fte-1/S3a protein could be also induced, purified tonsil B cells were infected with the B95.8 strain of EBV. In parallel experiments, tonsil B cells were treated with lipopolysaccharide (LPS; 100 mg/ml), a known activator of B cells. Cell activation by LPS was confirmed by CD150 stainings (data not shown). The cells were harvested after 48 h, and the cell lysates were analyzed by Western blotting. As control, freshly isolated and untreated tonsil B cells and 1-month-old LCL were used. The cell lysates were prepared from the same amount of cells, and the same amount of lysates was applied for Western blotting. We observed a clear induction of Fte-1/S3a in the freshly EBV-infected cells but not in the LPS-activated cells (Fig. 3A). The level of Fte-1/S3a remained high in the LCL. The Fte-1/S3a induction by EBV was observed also by immunostaining. Fte-1/S3a levels were increased dramatically in the EBV-infected, EBNA-5-positive cells (Fig. 3B).
Discussion EBNA-5 is a nuclear phosphoprotein composed of a variable number of the 66-amino acid long repeats (coded by the BamHI W1W2 repeats) flanked by a 44-amino acid long C-terminal unique region. Freshly infected B cells regularly express an entire ladder of EBNA-5 variants that are result of alternative splicing of variable number of repeats. All variants localize to the nucleus except those that only contain a single repeat and a unique region that remain in the cytoplasm [13]. In the nucleus, EBNA-5 is tightly associated with the nuclear matrix. It forms fine nuclear speckles in freshly infected B cells. At later phase of the infection and in established lymphoblastoid cell lines, LCLs, EBNA-5 is primarily found in the PML bodies [27,28]. This phenomenon is specific for the LCLs or B lymphoma cells with immunoblast phenotype. In any other cell types, exogenously introduced EBNA-5 localizes to finely distributed nuclear speckles [21]. Upon stress conditions (heat shock or proteasome inhibitor treatment) EBNA-5 moves to the nucleoli, where
it colocalizes with a number of cellular proteins, such as Hsp70, PML, 20S proteasome subunit, and mutant p53 [21]. We have previously observed that overexpression of the nucleolar p14ARF protein changed the localization of EBNA-5 [15]. EBNA-5 showed finely dispersed, homogeneous pattern in the transfected MCF7 cells but moved to the p14ARF-positive nuclear (extranucleolar) inclusions where these two proteins colocalized. We report now that EBNA-5 binds to and colocalizes with endogenous Fte-1/S3a within the nucleoli and in extranucleolar speckles. When Fte-1/S3a was overexpressed, it colocalized with the transfected EBNA-5 in extranucleolar precipitates. EBNA-5 is a protein required for efficient EBV-mediated immortalization. Deletion of its C-terminus decreases the viral-transforming activity [29]. One of the best characterized features of EBNA-5 is to enhance the EBNA-2regulated transcription (for review, see Ref. [30]). EBNA5 was implicated in the inhibition of pre-mRNA cleavage and polyadenylation [31]. However, the mechanism of the inhibition is not yet clarified. It was shown that none of EBNA-2-6 proteins could bind RNA or DNA. Probably, EBNA-5 may influence mRNA processing through the binding to proteins that control this process. The EBNA-5 mutants that contained one W1W2 repeat or only Y domain did not inhibit pre-mRNA cleavage and polyadenylation. Obviously, deleted EBNA-5 cannot fulfill its functions. Importantly, at least four W1W2 repeats and Y domain is needed to bind Fte-1/S3a protein. The full range of EBNA-5 function is not yet understood. A possible way to elucidate the function of EBNA-5 is to identify its cellular binding partners. As already mentioned, so far, 10 different possible EBNA-5 binding partners have been reported, including the chaperones Hsp70, Hsc70 [10], and Hsp27 [11]. Betatubulin, alpha-tubulin, and HA95 (A-kinase anchoring protein) were also identified as EBNA-5 binding partners [11]. Recently, HERR1 [14], HAX1 [12,13], 4P-alpha1[11], and p14ARF were found to bind EBNA-5. The homology between EBNA-5 binding proteins is very low (not more than 15%), except heat shock proteins. The Fte-1/ S3a protein is not homologue to any of the 10 EBNA-5 binding proteins. Fte-1 protein was identified as a protein that is needed for v-fos transformation of rat fibroblasts (Fte-1: v-fos transformation effector) [16]. Monoallelic disruption of Fte-1 gene was able to block v-fos-induced cell transformation. Reintroduction of exogenous Fte-1 cDNA into Fte-1 +/ transformation revertant clones restored the malignant phenotypes. The gene was localized to chromosome 4q31 [32,33]. The gene was also independently cloned as a TNF-alphainduced gene (TU-11) [34]. The Fte-1 gene was later shown to be identical to S3a ribosomal gene. S3a protein is part of the small subunit of ribosomes and is involved in various
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Fig. 3. (A) EBV induces Fte-1/S3a protein level. Western blot, probed with anti-Fte-1/S3a goat antibody. Line 1—nonactivated tonsil B cells; line 2—tonsil B cells, activated after treatment with LPS for 48 h; line 3—tonsil B cells, freshly infected with EBV (48 h); line 4—not loaded; line 5—freshly established (1 month) lymphoblastoid cell line (LCL). (B) EBV infection induces the Fte-1/S3a protein. Tonsil B cells infected with EBV at the 48 h after infection. Note the induction of endogenous Fte-1/S3a protein (red) in the EBV-infected, EBNA-5 (green)-positive cells. Blue—DNA staining.
steps of translation. Fte-1/S3a shows both cytoplasmic and nuclear (including nucleolar) localization. Fte-1/S3a mRNA and protein levels are increased in transformed cells [34] and in tumors such as colonic adenocarcinomas [35], feline thymic tumor [36], medullary thyroid carcinomas, especially in the metastases [37], hepatocellular carcinomas [38], DLBCL, and SIV-associated monkey lymphoma [39]. Mono-allelic disruption of the Fte-1/S3a gene altered polysome profiles and decreased the rate of protein
synthesis in the cells [40]. Fte-1/S3a was found to be highly up-regulated in the regenerating kidneys of partially nephrectomized mice [41]. Suppression of the Fte-1/S3a by antisense sequence led to the disruption of ovarian development in Drosophila melanogaster [42]. Importantly, it was shown that Fte-1/S3a protein levels are high in S phase of the cell cycle [40,43]. High Fte-1/S3a was found to bind to the transcription factor CHOP/ GADD153 that regulates erythroid differentiation. Overexpression of Fte-1/S3a inhibited differentiation of
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Rauscher cells induced either by erythropoietin or by dimethyl sulfoxide. This inhibition was reversed partially by simultaneous overexpression of CHOP or of antisense Fte-1/S3a [24]. Fte-1/S3a mRNA levels decreased significantly during neuroendocrine differentiation of human prostatic adenocarcinoma cells [44]. Fte-1/S3a was also found to associate with the automodification domain of poly(ADP-ribose) polymerase (PARP), an important apoptosis regulator [45]. S3a is one of the five ribosomal proteins that were phosphorylated after HSV1 (herpes simplex virus 1) infection [46]. Taken together, these data suggests that Fte-1/S3a plays an important role in regulation of cell cycle and proliferation by obviously performing extraribosomal functions. We observed a rise in Fte-1/S3a protein levels after EBV infection but not after LPS activation. This suggests that the increase of Fte-1/S3a level is associated with the virus-induced immortalization and not merely reflects the metabolic changes associated with B cell activation. The precise mechanism of EBV-induced Fte-1/S3a activation remains to be determined. Promoter studies in undifferentiated prostate adenocarcinoma cells suggested that CRE (cyclic AMP-responsive elements) were necessary for high level expression of the S3a gene [44]. Interestingly, the EBV Wp promoter that is responsible for the production of the first set of viral latency-associated mRNA is also regulated through CRE site [47]. Fte-1/ S3a may also be activated through LMP-1-mediated signaling that mimics constitutive activation of the TNF receptor family member CD40. Endogenous Fte-1/S3a protein is localized to the cytoplasm and to the nucleoli. We observed the enrichment of the endogeneous Fte-1/S3a protein in the nucleoli, when the cells were transfected with EBNA-5-expressing constructs. The two proteins, however, showed the highest level of colocalization at extranucleolar sites in the nuclei of transfected cells. Partial colocalization of endogenous EBNA-5 and S3a proteins was also observed in the nucleoli of the freshly infected cells (data not shown). EBNA-5 variants that contain multiple repeats are always nuclear. The single repeat variant that remains in the cytoplasm does not bind Fte-1/S3a. This suggests that binding of EBNA-5 may modify some of the ribosome-independent functions of Fte1/S3a but not the translation initiation-associated functions that are carried out in the cytoplasm. It would be important to study how Fte-1/S3a protein influences on the transformation process induced by EBV. EBNA-5 probably exerts influence on the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.
Acknowledgments We thank Mia Lfwbeer for the assistance with tonsil B cell isolation.
Cancerfonden and a matching grant from the Concern Foundation, Los Angeles, and the Cancer Research Institute, New York, supported this work. It was also supported by Svenska L7kars7llskapet, Svenska Institutet, and INTAS grant 011-2382.
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