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Journal of Virology, May 2005, p. 5499-5506, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5499-5506.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Modulation of the Cell Growth Regulator mTOR by Epstein-Barr Virus-Encoded LMP2A

Cary A. Moody,1,2 Rona S. Scott,1,2,4 Nazanin Amirghahari,3 Cherie-Ann Nathan,3,4 Lawrence S. Young,5 Chris W. Dawson,5 and John W. Sixbey1,2,4*

Center for Molecular and Tumor Virology,1 Department of Microbiology and Immunology,2 Department of Otolaryngology and Head and Neck Surgery,3 Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130,4 Cancer Research United Kingdom Institute for Cancer Studies, Birmingham B15-2TT, United Kingdom5

Received 3 May 2004/ Accepted 20 December 2004


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ABSTRACT
 
Control of translation initiation is one means by which cells regulate growth and proliferation, with components of the protein-synthesizing machinery having oncogenic potential. Expression of latency protein LMP2A by the human tumor virus Epstein-Barr virus (EBV) activates phosphatidylinositol 3-kinase/Akt located upstream of an essential mediator of growth signals, mTOR (mammalian target of rapamycin). We show that mTOR is activated by expression of LMP2A in carcinoma cells, leading to wortmannin- and rapamycin-sensitive inhibition of the negative regulator of translation, eukaryotic initiation factor 4E-binding protein 1, and increased c-Myc protein translation. Intervention by this DNA tumor virus in cellular translational controls is likely to be an integral component of EBV tumorigenesis.


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INTRODUCTION
 
Epstein-Barr virus (EBV) is an opportunistic pathogen that can manifest itself in human disease years after primary infection, specifically in lymphoid and epithelial cell malignancies of both immune-competent and immune-compromised hosts. EBV-associated cancers include Burkitt's lymphoma, Hodgkin's lymphoma, posttransplant lymphoproliferative disease, non-Hodgkin's lymphomas of AIDS, nasopharyngeal carcinoma, and gastric carcinoma (33). Despite its sporadic association with most tumors, EBV is deemed causal when present, based in part on the uniformity in every tumor cell of what in circulating peripheral blood lymphocytes is a variably reiterated EBV terminal repeat sequence (6, 32). Circularization of the viral genome within cells by random recombination of its terminal repeats produces fused ends of a length distinct for each infected cell. Homogeneity of the repeats in tumor cells provides a viral marker of clonality that implies EBV infection precedes cellular expansion and is thus factorial (26, 28).

Fused termini, which may vary by as many as 20 reiterations of the 500-bp repeat unit (6), comprise the first intron of an open reading frame that encodes EBV latency protein LMP2A (20, 36). LMP2A is a transmembrane protein with transforming potential in epithelial cells and is expressed in several EBV-associated malignancies (33, 38). In carcinoma cells infected in vitro, we previously showed that expression levels of LMP2A vary inversely with the size of intron 1, with episomes with the fewest terminal repeat units expressing the most LMP2A (23). In culture, cells maintaining episomes with the fewest terminal repeats became predominant (23). The selective advantage apparently conferred by LMP2A may relate to its activation of the phosphatidylinositol 3-kinase (PI3-kinase) and serine/threonine kinase Akt signaling pathway, which has a prominent role in cell proliferation and survival (24, 31, 38, 41).

The cellular kinase mammalian target of rapamycin (mTOR) has a central role in regulating cap-dependent translation initiation and has emerged as a principal mediator of cell growth and proliferation by its regulation of cellular machinery for protein synthesis (Fig. 1) (16). Activation of mTOR is mediated by several upstream signaling pathways, including PI3-kinase/Akt. Activated mTOR phosphorylates the downstream effectors eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP1) and p70 S6 kinase (S6K), critical components in a pathway regularly co-opted by viruses to ensure translation of their own mRNA (19, 37, 44). With the hypothesis that EBV monoclonality in carcinoma cells reflects positive selection for optimal LMP2A expression, we examined mTOR signaling as a downstream component of the LMP2A-activated PI3-kinase/Akt pathway that might support a process of EBV-driven cellular expansion.



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  		FIG. 1. Schematic of the mTOR pathway and the potential intersection with LMP2A-activated PI3-kinase/Akt signaling. Activation of mTOR by PI3-kinase/Akt regulates translation through two independent downstream effectors, S6K and eIF4E. mTOR phosphorylates and inactivates 4E-BP1, the repressor of cap-binding protein eIF4E, resulting in dissociation of eIF4E and increased translation initiation. mTOR phosphorylates and activates S6K, promoting ribosome biogenesis. S6K and eIF4E function independently to promote cell cycle progression and cell growth. Points at which rapamycin, wortmannin, or LY294002 inhibit the pathway are indicated. S6K, p70 S6K; ODC1, ornithine decarboxylase; HIF1{alpha}, hypoxia-inducible factor 1 alpha.


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MATERIALS AND METHODS
 
Cell culture and treatments. To generate LMP2A-expressing cells, the HONE-1 nasopharyngeal carcinoma cell line (46) (originally EBV positive but which lost EBV during passage) and the Ad/AH cell line (42) (a human adenocarcinoma of the nasopharynx) were stably transfected with the vector pZIPneo or pZIPneo containing the LMP2A open reading frame. LMP2-expressing polyclonal pools of each cell line were established under G418 selection. CCL.20.2, a conjunctival carcinoma cell line, was infected with EBVneor, a recombinant virus generated by insertion of a neomycin resistance cassette in the BDLF3 open reading frame of EBV strain Akata (5) (gift of L. Hutt-Fletcher). Single-cell clones of CCL20.2 were derived by cell sorting (FACSVantage SE; Becton Dickinson, San Jose, CA), and the episome copy number in each clone, together with the relative size of fused EBV DNA termini, was determined as previously described (23). The pattern of EBV latency gene expression in CCL20.2 clones (EBNA1 and LMP2A only) was previously characterized (23).

HONE-LMP2A, HONE-pZIPneo, Ad/AH-LMP2A, Ad/AH-pZIPneo, and CCL.20.2-EBV cells were maintained in Dulbecco's modified Eagle's medium (Cellgro; Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), glutamine, penicillin-streptomycin, and 400 µg of G418/ml (Mediatech, Herndon, VA). BL Bako (EBV positive) is a Burkitt's lymphoma-derived cell line that served as control. For treatment with the PI3-kinase inhibitor LY294002 (Promega, Madison, WI), cells were serum starved 5 h and then incubated in 50 µM LY294002 for 1 h; for wortmannin (Sigma, St. Louis, MO), cells were treated with 0.1 µM for 30 min; the mTOR inhibitor rapamycin (Calbiochem, La Jolla, CA) was used at doses from 1 to 100 ng/ml.

Antibodies. Immunoblot assays were performed with antibodies to phospho-mTOR (ser2448), mTOR, phospho-Akt (ser473), Akt, eIF4E, 4E-BP1 rabbit polyclonal, phospho-4EBP1 (Thr37/46), phospho-S6K (Thr389), S6K (Cell Signaling, Beverly, MA), and c-Myc (Santa Cruz, Santa Cruz, CA) and actin (Sigma), tubulin (Lab Vision, Fremont, CA), and LMP2A (antibody 14B7; gift from E. Kremmer) (12).

Preparation of lysates and immunoblotting. Whole-cell lysates were prepared using Laemmli buffer (0.5 M Tris HCl, 1% sodium dodecyl sulfate [SDS], bromophenol blue, 1 M sucrose). Lysates were boiled for 5 min and then stored at –80°C. Protein equivalent to 2 x 104 cells for each sample was separated on 10% SDS-polyacrylamide gel electrophoresis (PAGE) gels, except for analysis of mTOR (with 7.5%) and 4E-BP1 (with 12%). Proteins were transferred to Immobilon P membranes (Amersham, Piscataway, NJ). Membranes were incubated in blocking solution for 1 h at room temperature and then in appropriate primary antibody overnight at 4°C. Using a horseradish peroxidase-linked secondary antibody, proteins were visualized by enhanced chemiluminescence (ECL-Plus; Amersham). Densitometry to quantify protein was performed using Quantity One software. Experiments were performed a minimum of three times.

7-Methyl-GTP-Sepharose 4B pull-down assay. HONE-LMP2A cells were seeded in 100- by 20-mm dishes at 2.5 x 106 cells/dish. After 24 h, cells were cultured in the presence of 10 ng/ml rapamycin for the times indicated, washed once in cold phosphate-buffered saline (PBS), and harvested in lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (Cell Signaling). Protein concentration was determined using the Bio-Rad DC protein assay system (Bio-Rad, Hercules, CA). eIF4E was precipitated from 750 µg of total protein by incubating with 25 µl of 7-methyl-GTP-Sepharose 4B beads (Amersham) in a total volume of 1 ml of lysis buffer. After 24 h of incubation at 4°C, the beads were washed once in lysis buffer and twice in PBS. The beads were resuspended in a small volume of SDS-PAGE loading buffer and denatured at 100°C for 3 min. Precipitated proteins were separated on 12% SDS-PAGE gels and analyzed by immunoblotting for 4E-BP1 and eIF4E.

Metabolic labeling and immunoprecipitation of LMP2A. For 35S labeling of LMP2A protein, EBV-infected and uninfected clones of CCL20.2 and HONE-LMP2A cells were washed twice in PBS, incubated in 6 ml of methionine-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1 mM glutamine, and labeled for 16 h with 0.2 mCi of [Tran35S]methionine (ICN, Costa Mesa, CA). Following labeling, 2 x 106 cells were lysed in 500 µl of lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (Cell Signaling). Nuclei and insoluble material were removed by centrifugation. Cell lysates were precleared with 20 µl of protein A/G-Sepharose beads for 2 h at 4°C and then incubated with the LMP2A antibody 14B7 overnight. Washed beads were resuspended in 20 µl of SDS loading buffer, heated at 70°C for 5 min, and stored at –80°C until use. Precipitated proteins were separated on a 7.5% SDS-PAGE gel, which was subsequently dried and exposed to a phosphorimager screen.

RNA extraction, cDNA synthesis, and real-time PCR. RNA was extracted according to the RNA STAT60 protocol (Tel-test, Friendswood, TX). For cDNA synthesis, 5 µg of RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) by using random hexamer primers. Quantitative (real-time) reverse transcription-PCR (RT-PCR) to determine c-myc and LMP2A mRNA expression was performed using the TaqMan fluorogenic system (ABI Prism 7700 sequence detection system; PE Applied Biosystems, Foster City, CA) as previously described (23). The comparative cycle threshold (CT) method was used to assess differences in c-myc and LMP2A transcript levels, with CT values determined by automated threshold analysis. All samples were run in duplicate, together with reactions to quantify expression of an internal control gene, glyceraldehyde-3-phosphate dehydrogenase to normalize for any differences in the amount of total RNA added. Template- and RT-negative reactions served as controls. Forward and reverse primers to amplify c-myc mRNA were 5'-GGACGACGAGACCTTCATCAA-3' and 5'-CCAGCTTCTCTGAGACGAGCTT-3', with probe 5'-6-carboxyfluorescein (FAM)-AGAAGCCGCTCCACATACAGT-tetramethyl carboxyrhodamine (TAMRA)-3' (1). LMP2A forward and reverse primers were 5'-AGCGGGCAGAGGAAG-3' and 5'-AAGAGGTAGGGCGCAACAATT-3', with probe 5'-FAM-TCCAGTATGCCTGCCTG-TAMRA-3'. Experiments were performed in triplicate.


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RESULTS
 
LMP2A/PI3-kinase/Akt signaling cascade in carcinoma cells. We previously showed that epithelial cell clones containing EBV episomes with minimal terminal repeats became predominant in culture, with LMP2A levels varying inversely with terminal repeat units (23). To elucidate factors driving selection for high LMP2A expressers in an already transformed cell population, potential downstream effectors of LMP2A signaling were examined in two carcinoma cell lines stably transfected with LMP2A (Fig. 2A). Using the EBV-negative nasopharyngeal carcinoma cell line HONE-1 as a relevant genetic background for expression and function of latent EBV genes (46), we first confirmed LMP2A activation of the PI3-kinase/Akt signaling pathway previously shown to occur in normal human keratinocytes (24, 38). Although total levels of Akt protein were similar in LMP2A-expressing HONE-1 (HONE-LMP2A) cells and vector (HONE-pZIPneo) controls, the activated form phosphorylated on serine 473 was elevated in LMP2A-expressing cells (Fig. 2B) (2). Identical results were observed in the Ad/AH epithelial cell line, which stably expressed LMP2A. Wortmannin, a PI3-kinase inhibitor, blocked Akt phosphorylation in LMP2A-expressing cells, verifying that Akt activation is mediated through PI3-kinase in these cells (Fig. 2B).



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  		FIG. 2. LMP2A activates PI3-kinase/Akt in stably transfected cells. (A) Immunoblots for LMP2A in cell lysates of HONE-1 nasopharyngeal carcinoma cells and Ad/AH adenocarcinoma cells transfected with pZIP-LMP2A versus pZIPneo alone. Tubulin served as a loading control. (B) LMP2A expression is associated with increased levels of activated p-Akt (phosphorylated on serine 473). The PI3-kinase inhibitor wortmannin (0.1 µM for 30 min) inhibited Akt phosphorylation in all cells. Dimethyl sulfoxide (DMSO) was the vehicle control.

Activation of protein kinase mTOR. Akt affects multiple downstream targets which can be broadly grouped into two categories: factors affecting cell survival and cell death, and proteins controlling translation. LMP2A induces constitutive activation of PI3-kinase/Akt survival pathways in B lymphocytes, but in epithelial cells the effect is also on cell growth and proliferation (13, 24, 31, 38, 41). A downstream protein kinase, mTOR, has recently been implicated in PI3-kinase/Akt-dependent oncogenesis through its regulation of the cellular protein translation machinery (Fig. 1) (3).

Given the activation of the PI3-kinase/Akt signaling cascade in HONE-LMP2A and Ad/AH-LMP2A, we examined phosphorylation of mTOR at serine 2448 (Fig. 3). This is the residue targeted by Akt and a point of convergence of the PI3-kinase/Akt and mTOR signaling pathways (25), although the relevance of this phosphorylation to all mTOR functions remains unclear (16, 22). By densitometric analysis there was a 1.84-fold ± 0.07-fold (mean ± standard error of the mean [SEM]; n = 3) increase in phosphorylated mTOR in HONE-LMP2A relative to cells transfected with vector alone and a 1.6-fold ± 0.06-fold (n = 3) increase in Ad/AH-LMP2A relative to vector control (Fig. 3A). This increase in phosphorylation is equivalent to that observed upon physiologic stimulation of mTOR by insulin, a positive regulator of the mTOR pathway (25). Because these results obtained with transfectants were from bulk polyclonal pools of cells, they are unlikely to be due to clonal variation. Phosphorylated mTOR in HONE-LMP2A was sensitive to wortmannin (Fig. 3A), implicating PI3-kinase/Akt in the possible activation of mTOR in LMP2A-expressing cells. The continued presence of a basal level of p-mTOR despite wortmannin treatment reflects potential activation by pathways other than PI3-kinase/Akt. A similar effect of wortmannin was observed in the Ad/AH-LMP2A cell line (data not shown). LMP2A/PI3-kinase/Akt activation accompanied by increased mTOR (serine 2448) phosphorylation was likewise detected in EBV-infected CCL20.2 carcinoma cells, a more physiologically relevant system in which LMP2A expression is regulated within the framework of the whole genome (Fig. 3B).



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  		FIG. 3. LMP2A activates mTOR in a PI3-kinase/Akt-dependent manner. (A) Phosphorylation of mTOR at serine 2448 (p-mTOR), the residue targeted by Akt. p-mTOR was normalized to a loading control (actin), and the fold change relative to the vector (pZIPneo) control is indicated numerically. Wortmannin is a PI3-kinase inhibitor; dimethyl sulfoxide (DMSO) was the carrier control. A blot representative of three independent experiments is shown. (B) p-Akt and p-mTOR in EBV-infected, LMP2A-expressing CCL20.2 conjunctival carcinoma cells versus levels in the uninfected control. The fold increase in p-mTOR is indicated numerically, as determined by densitometry.

Phosphorylation of mTOR effectors 4E-BP1 and S6K. mTOR regulates cell growth and proliferation by phosphorylating (and activating) ribosomal S6K and by phosphorylating (and inactivating) 4E-BP1 (Fig. 1) (16, 22). Activated S6K stimulates translation of mRNAs with 5'-terminal oligopyrimidine tracts that code for components of the translational machinery, such as ribosomal proteins and translation elongation factors (18). mTOR-mediated phosphorylation of the translational repressor 4E-BP1 frees previously bound eIF4E, allowing it to form the multiprotein complex that binds to the 7-methyl-GTP cap structure at the 5' end of mRNA for translation initiation (15, 40). eIF4E is present in rate-limiting amounts relative to other components of the translational apparatus, making it a key regulatory translation factor.

Because the functional significance of mTOR phosphorylation at serine 2448 by Akt remains unclear, the status of downstream effectors S6K and 4E-BP1 is often used as the more definitive readout of mTOR activation (16). To determine if the mTOR phosphorylation observed was reflected in increased downstream activity, we first examined the status of S6K, whose phosphorylation at threonine 389 is central to activation (29). S6K phosphorylation was unremarkable in HONE-LMP2A and infected CCL20.2 cells (Fig. 4A), a finding which concurs with earlier reports in immortalized epithelial cells (38). It is of note that disparate activation of downstream effectors by mTOR has been reported in other systems as well (39). Moreover, it is the dysregulation of cap-dependent translation through alterations in the 4E-BP1/eIF4E pathway, not activation of S6K, that is most linked to human cancer (4), although activation of either S6K or 4E-BP1/eIF4E is sufficient to accelerate cell cycle progression (11, 34).



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  		FIG. 4. LMP2A-activated mTOR signals through downstream effectors 4E-BP1/eIF4E. (A) LMP2A/Akt/mTOR does not activate p70 S6K (S6K). Immunoblots show paired HONE-1 lysates and EBV-infected or -uninfected CCL20.2 probed with phospho-specific antibody to p-S6K(Ser 389) or antibody to S6K. Tubulin was the loading control. (B) Phosphorylation of 4E-BP1 in LMP2A-transfected HONE-1 and Ad/AH cells; {alpha} (unphosphorylated) and ß through {varepsilon} (increasingly phosphorylated) isoforms were detected on immunoblots with 4E-BP1-specific antibody and also with a phospho-specific antibody to p-4E-BP1(Thr37/46). eIF4E, whose activity is inhibited by sequestration to unphosphorylated 4E-BP1, was expressed equally in LMP2A-expressing cells and vector (pZIPneo) control. Actin served as a loading control. (C) Phosphorylation of 4E-BP1 in EBV-infected versus uninfected CCL20.2 carcinoma cells, consistent with mTOR activation (Fig. 3B). (D) LMP2A-induced phosphorylation of 4E-BP1 is wortmannin, LY294002, and rapamycin sensitive. Immunoblots are of cell lysates from HONE-LMP2A cells cultured in the presence of dimethyl sulfoxide (DMSO; vehicle control), rapamycin (10 or 100 ng/ml for 24 h), LY294002 (50 µM for 1 h), or wortmannin (0.1 µM for 30 min). The phosphorylated isoforms of 4E-BP1 (seen in the DMSO control) exhibit decreased electrophoretic mobility compared to hypophosphorylated isoforms induced by rapamycin, wortmannin, or LY294002.

In the case of the second downstream effector, 4E-BP1, sequential phosphorylation produces multiple 4E-BP1 isoforms that can be discriminated on immunoblotting by their electrophoretic mobilities (15). In HONE-LMP2A cells, 4E-BP1 was resolved into at least four isoforms by antibody to total 4E-BP1, the {alpha} isoform being unphosphorylated and most mobile and the ß, {gamma}, {delta}, and {varepsilon} isoforms being progressively more phosphorylated (Fig. 4B). By contrast, cells transfected with vector alone contained predominantly the hypophosphorylated {alpha} and ß isoforms. Identical findings were obtained in the second epithelial cell line, Ad/AH (Fig. 4B). Similarly, with a phospho-specific antibody to threonine residues 37/46 targeted by mTOR and which function as priming sites for sequential phosphorylations (14), 4E-BP1 was more abundantly phosphorylated in HONE-LMP2A and Ad/AH-LMP2A cells than in vector controls (Fig. 4B). Hyperphosphorylation of 4E-BP1 was likewise found in EBV-infected CCL20.2 cells that expressed LMP2A in the context of virus (Fig. 4C).

As further corroboration of mTOR's role in 4E-BP1 phosphorylation in LMP2A-expressing cells, we treated HONE-LMP2A cells with the immunosuppressive macrolide antibiotic rapamycin, a specific inhibitor of mTOR (Fig. 4D). Rapamycin reduced hyperphosphorylated isoforms of 4E-BP1 to levels comparable to those in untreated HONE-pZIPneo cells (Fig. 4B). Exposure of HONE-LMP2A cells to the PI3-kinase inhibitors wortmannin and LY294002 likewise shifted 4E-BP1 to predominantly the hypophosphorylated isoforms, suggesting involvement of LMP2A/PI3-kinase/Akt signaling upstream of the activated mTOR pathway (Fig. 4D).

4E-BP1/eIF4E dissociation in LMP2A-expressing cells. The hyperphosphorylation of 4E-BP1 observed in the LMP2A-expressing cells (Fig. 4B) suggests that eIF4E may be uncoupled from its repressor in these cells (Fig. 1). To show the status of eIF4E in HONE-LMP2A, where 4E-BP1 was hyperphosphorylated, we evaluated 4E-BP1/eIF4E association over a 24-hour time course exposure to rapamycin (Fig. 5). By allowing eIF4E to bind a 7-methyl-GTP cap analogue coupled to Sepharose beads, its associated proteins can be isolated and defined on Western analysis. Binding of 4E-BP1 to eIF4E does not inhibit its ability to bind to the cap but impairs the formation of a translationally active eIF4E multiprotein complex. At time zero, when 4E-BP1 is hyperphosphorylated in HONE-LMP2A, little of the repressor should be bound to eIF4E. Rapamycin treatment induced a shift from hyper- to hypophosphorylated isoforms of 4E-BP1 that was maximal at 2 h. Accordingly, by the 7-methyl-GTP-Sepharose pull-down assay eIF4E was largely uncoupled from 4E-BP1 at time zero, indicating that eIF4E is free to initiate translation in LMP2A-expressing cells. With rapamycin treatment, a maximal association of eIF4E to the newly hypophosphorylated 4E-BP1 isoforms occurred after 2 h of drug exposure (Fig. 5).



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  		FIG. 5. eIF4E is dissociated from phosphorylated 4E-BP1 in HONE-LMP2A cells. (Top) Immunoblot showing changing isoforms of 4E-BP1 in HONE-LMP2A cells during a time course exposure to rapamycin, with maximal phosphorylation at time zero and hypophosphorylation peaking at 2 h. (Bottom) Precipitation of eIF4E by 7-methyl-GTP-Sepharose beads, with precipitate immunoblotted for eIF4E and 4E-BP1. 4EBP1/eIF4E assembly was maximal coincident with the appearance of hypophosphorylated 4E-BP1.

Free eIF4E and protein synthesis. Because eIF4E levels were equivalent in HONE-LMP2A and -pZIPneo cells (Fig. 4B), differences in translational initiation would largely be determined by the relative proportions dissociated from repressor 4E-BP1 shown above. Phosphorylation of 4E-BP1 by mTOR, with release of bound eIF4E, facilitates the selective translation of mRNAs with complex 5' secondary structures, many of which code for growth-related proteins such as c-Myc, vascular endothelial growth factor, fibroblast growth factor, cyclin D1, hypoxia-inducible factor 1 alpha, and ornithine decarboxylase (10, 35).

To determine the impact of LMP2A on translation in carcinoma cell lines, we examined levels of c-Myc protein in HONE-LMP2A and Ad/AH-LMP2A cells versus vector controls (Fig. 6A). On immunoblots, c-Myc protein was increased by greater than twofold in both LMP2A-expressing cell lines (Fig. 6A). Furthermore, when HONE-LMP2A and Ad/AH-LMP2A cells were treated with rapamycin, c-Myc protein reverted to levels comparable to vector controls (Fig. 6A). To ensure that the differences in protein level did not reflect variation in transcription, mRNA levels were determined by quantitative real-time RT-PCR (Fig. 6B). mRNA levels of c-myc were not increased in LMP2A-expressing cells relative to vector control, confirming a transcription-independent elevation in c-Myc protein, consistent with mTOR-enhanced translation.



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  		FIG. 6. LMP2A increases c-Myc expression in a rapamycin-sensitive manner. (A) Immunoblot of c-Myc protein in HONE-LMP2A and Ad/AH-LMP2A cells versus vector (pZIPneo) controls. Cells were treated for 24 h with the inhibitor of mTOR, rapamycin (100 ng/ml), or dimethyl sulfoxide (DMSO; vehicle control). Numerical values indicate the fold change in c-Myc relative to the pZIPneo control after normalization to the actin loading control. Shown is a representative of three independent experiments. (B) c-Myc elevation is transcription independent. Results shown are the relative fold difference in c-myc transcript levels as determined by quantitative real-time PCR analysis of total RNA from HONE-LMP2A and Ad/AH-LMP2A cells versus pZIPneo vector controls. As in panel A above, cells were treated with rapamycin or DMSO. Transcript levels of c-myc were normalized to HONE-LMP2A (or Ad/AH-LMP2A) levels, which were set at 1. Shown are values from three independent experiments, with error bars representing the SEM.

EBV terminal repeat units, level of LMP2A expression, and differential mTOR signaling in infected carcinoma cells. In EBV-infected CCL20.2 epithelial cells which expressed EBV latency proteins EBNA1 and LMP2A only, both LMP2A expression and cell population doubling times were shown previously to vary inversely with EBV terminal repeat number (23). To determine if differential growth in these cells might reflect disproportionate signaling via the PI3-kinase/Akt/mTOR pathway, we examined two previously characterized CCL20.2 clones bearing an equivalent number of EBV episomes but with approximately 4 versus 20 terminal repeats (23). Quantitative analysis of LMP2A at the mRNA level showed an approximate fourfold difference in expression between the EBV-infected subclones (Fig. 7A). Despite the low level of LMP2A transcripts in the two CCL20.2 clones relative to HONE-LMP2A and an EBV-positive Burkitt's lymphoma cell control (Fig. 7A), LMP2A protein levels in those cells, as detected by immunoprecipitation of [35S]methionine-labeled LMP2A, were proportionate to mRNA differences (Fig. 7B).



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  		FIG. 7. Effect of EBV terminal repeat number on LMP2A expression and signaling in EBV-infected CCL20.2 carcinoma cell clones. (A) Relative LMP2A transcript levels in transfected HONE-LMP2A and EBV-infected CCL20.2 clones 25 and 30 (each having 3 episomes/cell but with 4 versus 20 terminal repeats, respectively). Levels were calculated as a percentage of LMP2A expression in the EBV-positive Burkitt's lymphoma cell line Bako (45 episomes/cell). Quantification by real-time RT-PCR using the TaqMan fluorogenic system has been previously described (23). (B) Differential LMP2A signaling to translational effectors in EBV-infected CCL20.2 clone 25 (episomes with 4 terminal repeats), clone 30 (episomes with 20 terminal repeats), and uninfected parental cells. LMP2A protein, relative to transcript level in panel A above, is shown by [35S]methionine labeling and immunoprecipitation with anti-LMP2A antibody 14B7. Clone 25 shows increased p-Akt, phosphorylated 4E-BP1 (antibodies to both total 4E-BP1 and phospho-specific antibody to p-4E-BP1 Thr 37/46), and c-Myc protein relative to clone 30 and uninfected parental cells. The fold increase in c-Myc (normalized to tubulin) is indicated, relative to EBV-negative CCL20.2 set at 1.

In immunoblot assays, phosphorylated Akt was higher in the CCL20.2 clone with the fewest repeats and greatest LMP2A expression (Fig. 7B). Likewise, the downstream repressor of mRNA translation, 4E-BP1, was hyperphosphorylated to a greater extent in the clone with only four terminal repeats (Fig. 7B), reflecting activated mTOR in those cells (Fig. 3B). Consistent with release of bound eIF4E from phosphorylated 4E-BP1 and enhanced translation initiation, c-Myc protein was increased in the clone with the least terminal repeats (Fig. 7B). These results suggest that, when expressed at physiologic levels in the context of genomes with a variable terminal repeat number, LMP2A activates the mTOR signaling cascade in a graded fashion that may promote clonal epithelial cell outgrowth.


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DISCUSSION
 
mTOR is a key downstream mediator in the insulin-like growth factor I receptor/PI3-kinase/Akt signaling pathway that regulates cell proliferation, growth differentiation, migration, and survival (22, 27, 43). Elevated levels or constitutive activation of PI3-kinase, Akt, and mTOR signaling components have been detected in numerous human cancers, including lymphomas, melanomas, central nervous system malignancies, and carcinomas of the head and neck, lung, bladder, kidney, ovary, prostate, breast, and pancreas (10, 43). Oncogenic transformation consequent to PI3-kinase and Akt gain of function can be inhibited by rapamycin, whereas transforming activities of other oncoproteins are unaffected by the drug (3, 17).

LMP2A activation of mTOR growth regulatory pathways as described here offers several important insights into the association of EBV with human cancers. Cast largely as a tumor initiator based on evidence for viral clonality in malignant tissue, EBV may also serve as a critical factor in tumor progression. One could speculate that fortuitous infection of an evolving malignancy by virus endogenous to the host, with subsequent LMP2A-driven progression of tumors from a polyclonal to monoclonal viral genotype, might explain not only the inconsistent association of EBV with tumors as diverse as carcinomas, leiomyosarcomas, and hematological malignancies but also the outcome of EBV clonality. Such a scenario would more fully acknowledge viral opportunism in the context of the life-long carrier state, with infection facilitated by genetic and epigenetic alterations intrinsic to cancer cells (7-9). Establishment of a greater protein synthesis output, in this case through inactivation of the translational inhibitor 4E-BP1, may be a required step in the progression of malignancies, allowing cancer cells to sustain their proliferative state or enhance survival by increasing translational efficiencies of oncogene transcripts (10). The apparent greater activation of Akt/mTOR signaling that we have shown in epithelial cells bearing EBV genomes with limited terminal repeats confirms LMP2A functionality at levels detected in this study (Fig. 7) and corroborates the notion of clonal emergence based on differential LMP2A expression (23).

These findings portray what is likely to be a seamless integration of the machinery of protein synthesis with transcriptional alterations in cell cycle genes accompanying EBV growth transformation (30). In the case of LMP2A/PI3-kinase/Akt signaling in epithelial cells, not only is mTOR's regulation of protein translation altered as described here, but the Wnt/ß-catenin pathway is also engaged via the Akt downstream effector glycogen synthase kinase 3 (24). ß-Catenin activates members of the T-cell factor/lymphoid enhancer factor family of transcription factors that increase expression of growth-promoting genes, encoding the very class of mRNAs with a high degree of secondary structure in their 5' untranslated region that heavily depend on eIF4E for translation initiation (10, 35, 40). Such convergence of signaling pathways, meshing the transcription of growth-related genes with the means for their selective translation, may provide potent strategies for treatment of EBV-associated diseases (4, 17, 21, 45).


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ACKNOWLEDGMENTS
 
We thank D. Duhon for technical assistance and J. Cardelli, A. Yurochko, E. Kremmer, and L Hutt-Fletcher for reagents.

This work was supported by Public Health Service grants CA67372 from the National Cancer Institute and P20RR18724 from the National Center for Research Resources.


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FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130. Phone: (318) 675-4272. Fax: (318) 675-5764. E-mail: jsixbe{at}lsuhsc.edu. Back


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REFERENCES
 
    1
  1. Akervall, J., U. Bockmühl, I. Petersen, K. Yang, T. E. Carey, and D. M. Kurnit. 2003. The gene ratios c-MYC:cyclin-dependent kinase (CDK)N2A and CCND1:CDKN2A correlate with poor prognosis in squamous cell carcinoma of the head and neck. Clin. Cancer Res. 9:1750-1755.[Abstract/Free Full Text]
    2. 2
  2. Alessi, D. R., M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, and B. A. Hemmings. 1996. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15:6541-6551.[Medline]
    3. 3
  3. Aoki, M., E. Blazek, and P. K. Vogt. 2001. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt. Proc. Natl. Acad. Sci. USA 98:136-141.[Abstract/Free Full Text]
    4. 4
  4. Bjornsti, M. A., and P. J. Houghton. 2004. Lost in translation: dysregulation of cap-dependent translation and cancer. Cancer Cell 5:519-523.[CrossRef][Medline]
    5. 5
  5. Borza, C. M., and L. M. Hutt-Fletcher. 1998. Epstein-Barr virus recombinant lacking expression of glycoprotein gp150 infects B cells normally but is enhanced for infection of epithelial cells. J. Virol. 72:7577-7582.[Abstract/Free Full Text]
    6. 6
  6. Brown, N. A., C.-R Liu, Y.-F. Wang, and C. R. Garcia. 1988. B-cell lymphoproliferation and lymphomagenesis are associated with clonotypic intracellular terminal regions of the Epstein-Barr virus. J. Virol. 62:962-969.[Abstract/Free Full Text]
    7. 7
  7. Chan, A. S. C., K. F. To, K. W. Lo, K. F. Mak, W. Pak, B. Chiu, G. M. K. Tse, M. Ding, X. Li, J. C. K. Lee, and D. P. Huang. 2000. High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from southern Chinese. Cancer Res. 60:5365-5370.[Abstract/Free Full Text]
    8. 8
  8. Chan, A. S. C., K. F. To, K. W. Lo, M. Ding, X. Li, P. Johnson, and D. P. Huang. 2002. Frequent chromosome 9p losses in histologically normal nasopharyngeal epithelia from southern Chinese. Int. J. Cancer 102:300-303.[CrossRef][Medline]
    9. 9
  9. Chen, H., J. M. Lee, Y. Zong, M. Borowitz, M. H. Ng, R. F. Ambinder, and S. D. Hayward. 2001. Linkage between STAT regulation and Epstein-Barr virus gene expression in tumors. J. Virol. 75:2929-2937.[Abstract/Free Full Text]
    10. 10
  10. De Benedetti, A., and A. L. Harris. 1999. eIF4E expression in tumors: its possible role in progression of malignancies. Int. J. Biochem. Cell Biol. 31:59-72.[CrossRef][Medline]
    11. 11
  11. Fingar, D. C., C. J. Richardson, A. R. Tee, L. Cheatham, C. Tsou, and J. Blenis. 2004. mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol. Cell. Biol. 24:200-216.[Abstract/Free Full Text]
    12. 12
  12. Fruehling, S., S. K. Lee, R. Herrold, B. Frech, G. Laux, E. Kremmer, F. A. Grasser, and R. Longnecker. 1996. Identification of latent membrane protein 2A (LMP2A) domains essential for the LMP2A dominant-negative effect on B-lymphocyte surface immunoglobulin signal transduction. J. Virol. 70:6216-6226.[Abstract]
    13. 13
  13. Fukuda, M., and R. Longnecker. 2004. Latent membrane protein 2A inhibits transforming growth factor-ß1-induced apoptosis through the phosphatidylinositol 3-kinase/Akt pathway. J. Virol. 78:1697-1705.[Abstract/Free Full Text]
    14. 14
  14. Gingras, A. C., S. P. Gygi, B. Raught, R. D. Polakiewicz, R. T. Abraham, M. F. Hoekstra, R. Aebersold, and N. Sonenberg. 1999. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev. 13:1422-1437.[Abstract/Free Full Text]
    15. 15
  15. Gingras, A. C., S. G. Kennedy, M. A. O'Leary, N. Sonenberg, and N. Hay. 1998. 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev. 12:502-513.[Abstract/Free Full Text]
    16. 16
  16. Hay, N., and N. Sonenberg. 2004. Upstream and downstream of mTOR. Genes Dev. 18:1926-1945.[Abstract/Free Full Text]
    17. 17
  17. Houghton, P. J., and S. Huang. 2004. mTOR as a target for cancer therapy. Curr. Top. Microbiol. Immunol. 279:339-359.[Medline]
    18. 18
  18. Jefferies, H. B., S. Fumagalli, P. B. Dennis, C. Reinhard, R. B. Pearson, and G. Thomas. 1997. Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. EMBO J. 16:3693-3704.[CrossRef][Medline]
    19. 19
  19. Kudchodkar, S. B., Y. Yu, T. G. Maguire, and J. C. Alwine. 2004. Human cytomegalovirus infection induces rapamycin-insensitive phosphorylation of downstream effectors of mTOR kinase. 78:11030-11039.
    20. 20
  20. Laux, G., M. Perricaudet, and P. J. Farrell. 1988. A spliced Epstein-Barr virus gene expressed in immortalized lymphocytes is created by circularization of the linear genome. EMBO J. 7:769-774.[Medline]
    21. 21
  21. Majewski, M., M. Korecka, P. Kossev, S. Li, J. Goldman, J. Moore, L. E. Silberstein, P. C. Nowell, W. Schuler, L. M. Shaw, and M. A. Wasik. 2000. The immunosuppressive macrolide RAD inhibits growth of human Epstein-Barr virus-transformed B lymphocytes in vitro and in vivo: a potential approach to prevention and treatment of posttransplant lymphoproliferative disorders. Proc. Natl. Acad. Sci. USA 97:4285-4290.[Abstract/Free Full Text]
    22. 22
  22. Martin, K. A., and J. Blenis. 2002. Coordinate regulation of translation by the PI3-kinase and mTOR pathways. Adv. Cancer Res. 86:1-39.[Medline]
    23. 23
  23. Moody, C. A., R. S. Scott, T. Su, and J. W. Sixbey. 2003. Length of Epstein-Barr virus termini as a determinant of epithelial cell clonal emergence. J. Virol. 77:8555-8561.[Abstract/Free Full Text]
    24. 24
  24. Morrison, J. A., A. J. Klingelhutz, and N. Raab-Traub. 2003. Epstein-Barr virus latent membrane protein 2A activates ß-catenin signaling in epithelial cells. J. Virol. 77:12276-12284.[Abstract/Free Full Text]
    25. 25
  25. Nave, B. T., M. Ouwens, D. J. Withers, D. R. Alessi, and P. R. Shepherd. 1999. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem. J. 344:427-431.
    26. 26
  26. Neri, A., F. Barriga, G. Inghirami, D. M. Knowles, J. Neequaye, I. T. Magrath, and R. Dalla-Favera. 1991. Epstein-Barr virus infection precedes clonal expansion in Burkitt's and acquired immunodeficiency syndrome-associated lymphoma. Blood 77:1092-1095.[Abstract/Free Full Text]
    27. 27
  27. Oldham, S., and E. Hafen. 2003. Insulin/IGF and target of rapamycin signaling: a TOR de force in growth control. Trends Cell Biol. 13:79-85.[CrossRef][Medline]
    28. 28
  28. Pathmanathan, R., U. Prasad, R. Sadler, K. Flynn, and N. Raab-Traub. 1995. Clonal proliferations of cells infected with Epstein-Barr virus in pre-invasive lesions related to nasopharyngeal carcinoma. N. Engl. J. Med. 333:693-698.[Abstract/Free Full Text]
    29. 29
  29. Pearson, R. B., P. B. Dennis, J. W. Han, N. A. Williamson, S. C. Kozma, R. E. Wittenhall, and G. Thomas. 1995. The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J. 14:5279-5287.[Medline]
    30. 30
  30. Portis, T., P. Dyck, and R. Longnecker. 2003. Epstein-Barr virus (EBV) LMP2A induces alterations in gene transcription similar to those observed in Reed-Sternberg cells of Hodgkin lymphoma. Blood 102:4166-4178.[Abstract/Free Full Text]
    31. 31
  31. Portis, T., and R. Longnecker. 2004. Epstein-Barr virus (EBV) LMP2A mediates B-lymphocyte survival through constitutive activation of the Ras/PI3K/Akt pathway. Oncogene 23:8619-8628.[CrossRef][Medline]
    32. 32
  32. Raab-Traub, N., and K. Flynn. 1986. The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell 47:883-889.[CrossRef][Medline]
    33. 33
  33. Rickinson, A. B., and E. Kieff. 2001. Epstein-Barr virus, p. 2575-2627. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Strauss (ed.), Field's virology, 4th ed. Lippincott Williams & Wilkins, Philadelphia, Pa.
    34. 34
  34. Rousseau, D., A. C. Gingras, A. Pause, and N. Sonenberg. 1996. The eIF4E-binding proteins 1 and 2 are negative regulators of cell growth. Oncogene 13:2415-2420.[Medline]
    35. 35
  35. Rousseau, D., R. Kaspar, I. Rosenwald, L. Gehrke, and N. Sonenberg. 1996. Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E. Proc. Natl. Acad. Sci. USA 93:1065-1070.[Abstract/Free Full Text]
    36. 36
  36. Sample, J., D. Liebowitz, and E. Kieff. 1989. Two related Epstein-Barr virus membrane proteins are encoded by separate genes. J. Virol. 63:933-937.[Abstract/Free Full Text]
    37. 37
  37. Schneider, R. J., and I. Mohr. 2003. Translation initiation and viral tricks. Trends Biochem. Sci. 28:130-136.[CrossRef][Medline]
    38. 38
  38. Scholle, F., K. M. Bendt, and N. Raab-Traub. 2000. Epstein-Barr virus LMP2A transforms epithelial cells, inhibits cell differentiation, and activates Akt. J. Virol. 74:10681-10689.[Abstract/Free Full Text]
    39. 39
  39. Shi, Y., J.-H. Hsu, L. Hu, J. Gera, and A. Lichtenstein. 2002. Signal pathways involved in activation of p70S6K and phosphorylation of 4E-BP1 following exposure of multiple myeloma tumor cells to interleukin-6. J. Biol. Chem. 277:15712-15720.[Abstract/Free Full Text]
    40. 40
  40. Sonenberg, N., and A. C. Gingras. 1998. The mRNA 5' cap-binding protein eIF4E and control of cell growth. Curr. Opin. Cell Biol. 10:268-275.[CrossRef][Medline]
    41. 41
  41. Swart, R., I. K. Ruf, J. Sample, and R. Longnecker. 2000. Latent membrane protein 2A-mediated effects on the phosphatidylinositol 3-kinase/Akt pathway. J. Virol. 74:10838-10845.[Abstract/Free Full Text]
    42. 42
  42. Takimoto, T., M. Furukawa, M. Hatano, and R. Umeda. 1984. Epstein-Barr virus nuclear antigen-positive nasopharyngeal hybrid cells. Ann. Otol. Rhinol. Laryngol. 93:166-169.[Medline]
    43. 43
  43. Vogt, P. K. 2001. PI3-kinase, mTOR, protein synthesis and cancer. Trends Mol. Med. 7:482-484.[CrossRef][Medline]
    44. 44
  44. Walsh, D., and I. Mohr. 2004. Phosphorylation of eIF4E by Mnk-1 enhances HSV-1 translation and replication in quiescent cells. Genes Dev. 18:660-672.[Abstract/Free Full Text]
    45. 45
  45. Wendel, H.-G., E. de Stanchina, J. S. Fridman, A. Malina, S. Ray, S. Kogan, C. Cordon-Cardo, J. Pelletier, and S. W. Lowe. 2004. Survival signaling by Akt and eIF4E in oncogenesis and cancer therapy. Nature 428:332-337.[CrossRef][Medline]
    46. 46
  46. Yao, K. T., H. Y. Zhang, H. C. Zhu, F. X. Wang, G. Y. Li, D. S. Wen, Y. P. Li, C. H. Tsai, and R. Glaser. 1990. Establishment and characterization of two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus and derived from nasopharyngeal carcinomas. Int. J. Cancer 45:83-89.[Medline]

Journal of Virology, May 2005, p. 5499-5506, Vol. 79, No. 9
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.9.5499-5506.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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