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Originally published In Press as doi:10.1074/jbc.M304771200 on October 7, 2003

J. Biol. Chem., Vol. 278, Issue 51, 51134-51142, December 19, 2003
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Latent Membrane Protein 1 of Epstein-Barr Virus Stimulates Processing of NF-{kappa}B2 p100 to p52*

Peter G. P. Atkinson{ddagger}§, Helen J. Coope{ddagger}§, Martin Rowe¶, and Steven C. Ley{ddagger}||

From the {ddagger}Division of Immune Cell Biology, National Institute for Medical Research, Mill Hill, London, NW7 1AA and Section of Infection and Immunity, University of Wales College of Medicine, Cardiff, CF4 4XX, United Kingdom

Received for publication, May 7, 2003 , and in revised form, September 16, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have identified a limited number of cellular receptors that can stimulate an alternative NF-{kappa}B activation pathway that depends upon the inducible processing of NF-{kappa}B2 p100 to p52. Here it is shown that the latent membrane protein (LMP)-1 of Epstein-Barr virus can trigger this signaling pathway in both B cells and epithelial cells. LMP1-induced p100 processing, which is mediated by the proteasome and is dependent upon de novo protein synthesis, results in the nuclear translocation of p52·RelB dimers. Previous studies have established that LMP1 also stimulates the canonical NF-{kappa}B-signaling pathway that triggers phosphorylation and degradation of I{kappa}B{alpha}. Interestingly, LMP1 activation of these two NF-{kappa}B pathways is shown here to require distinct regions of the LMP1 C-terminal cytoplasmic tail. Thus, C-terminal-activating region 1 is required for maximal triggering of p100 processing but is largely dispensable for stimulation of I{kappa}B{alpha} phosphorylation. In contrast, C-terminal-activating region 2 is critical for maximal LMP1 triggering of I{kappa}B{alpha} phosphorylation and up-regulation of p100 levels but does not contribute to activation of p100 processing. Because p100 deletion mutants that constitutively produce p52 oncogenically transform fibroblasts in vitro, it is likely that stimulation of p100 processing by LMP1 will play an important role in its transforming function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epstein-Barr virus (EBV)1 is a ubiquitous human herpes virus that infects B-lymphocytes and certain epithelial cells (1-3). EBV is the causative agent of infectious mononucleosis and is also implicated in the etiology of both epithelial (nasopharyngeal carcinoma) and lymphoid malignancies (Burkitt's lymphoma and Hodgkin's disease). Infection of primary human B lymphocytes in vitro with EBV leads to their immortalization and the establishment of lymphoblastoid cell lines (LCLs). Latent membrane protein (LMP) 1 is one of five latent genes shown to be essential for EBV-induced transformation of B cells (4). LMP1 is required for both LCL establishment and continued proliferation (4-6). The transforming potential of LMP1 has been confirmed both in vitro in fibroblast transformation assays (7) and in vivo in transgenic mice (8). Significantly, LMP1 is expressed in the majority of EBV-associated human malignancies (1).

LMP1 is an integral membrane protein with six hydrophobic transmembrane domains that mediate its constitutive oligomerization and targeting to plasma membrane lipid rafts (9). Constitutive oligomerization allows LMP1 to function as a ligand-independent receptor and is essential for its transforming potential in both fibroblasts and B cells. The 200-amino acid cytoplasmic C terminus of LMP1 is also required for cell transformation (10). This contains two subdomains, termed C-terminal-activating regions (CTARs) 1 and 2, which act as docking sites for complexes of signaling proteins that trigger activation of the transcription factors NF-{kappa}B and activator protein-1 (10). NF-{kappa}B activation is absolutely required to block apoptosis of EBV-transformed LCLs (11) and is also required for LMP1 transformation of fibroblasts (12).

Mammalian cells express five NF-{kappa}B proteins: RelA, RelB, c-Rel, NF-{kappa}B1 p50, and NF-{kappa}B2 p52, which combine to form homo- and heterodimers that regulate genes involved in immune responses, apoptosis, and development (13). NF-{kappa}B dimers are retained in the cytoplasm of unstimulated cells by interaction with a family of inhibitory proteins (I{kappa}Bs), which includes I{kappa}B{alpha}. Activation of the canonical NF-{kappa}B-signaling pathway by agonists such as tumor necrosis factor (TNF) {alpha} and interleukin 1 induces I{kappa}B{alpha} phosphorylation, ubiquitination, and subsequent proteolysis by the proteasome. NF-{kappa}B dimers, which are predominantly p50-RelA heterodimers, are thereby released to translocate into the nucleus and modulate gene expression. Signal-induced phosphorylation of I{kappa}B{alpha} is mediated by the I{kappa}B kinase (IKK) complex, which comprises two catalytic subunits, IKK1 (IKK{alpha}) and IKK2 (IKK{beta}), and a structural subunit NEMO (IKK{gamma}) (14, 15). Genetic studies indicate that IKK2 is essential for I{kappa}B{alpha} phosphorylation triggered by TNF{alpha} or interleukin 1, whereas IKK1 is largely dispensable (15).

Recently, an "alternative" NF-{kappa}B activation pathway has been described (16). This pathway triggers proteasome-mediated processing of the NF-{kappa}B2 precursor p100 to produce p52. In contrast to the canonical NF-{kappa}B pathway, which is triggered by multiple different stimuli (15), only three cellular receptors have so far been shown to stimulate p100 processing; namely, B cell-activating factor receptor (17, 18), lymphotoxin {beta} receptor (LT{beta}-R), (19) and CD40 (20). Receptor activation of this pathway is critically dependent on the mitogen-activated protein 3-kinase NIK and IKK1 (21, 22).

Functionally, LMP1 resembles an activated CD40 receptor, with both molecules promoting B cell survival and proliferation and regulating a highly overlapping spectrum of activation markers (23). Indeed, activation of CD40 can compensate for the lack of LMP1 expression and promote short term growth of EBV-transformed LCLs (6, 24). It is firmly established that LMP1 activates the canonical NF-{kappa}B pathway that regulates I{kappa}B{alpha} proteolysis (9). In the present study evidence is presented that in B cells and 293 epithelial cells LMP1 also induces NF-{kappa}B2 p100 processing to p52, similar to CD40 (20). The potential importance of the alternative NF-{kappa}B signaling pathway for cell transformation by LMP1 is discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
cDNA Constructs, Antibodies, and Reagents—The following plasmids have been described previously: pSG5-tCD2 (tCD2, extracellular and transmembrane domains of rat CD2), pSG5-CD2-LMP 192, pSG5-CD2-LMP1 (25), pcDNA3-Myc-p100, pcDNA3-p100S866A,S870A, pcDNA3-Myc-p100{Delta}GRR (residues 346-377 deleted), and pcDNA3-Myc-NIK{Delta}N (residues 624-947 of NIK) (20). The tetracycline-regulated pJEF vector constructs encoding EBNA1, EBNA2, LMP1, LMP2 (26), and EBNA LP (27) have been described elsewhere. The following plasmids were generous gifts from the originating laboratories: pSV-LMP1, pSV-LMP1Y384G, pSV-LMP1AAA (residues P204A,Q206A,T208A), pSV-LMP1AAA/Y384G (28), pCMV HA-ubiquitin (29), pCR-3-FLAG IKK1S176A,S180A (IKK1.dn), pCR-3-FLAG IKK2S177A,S181A (IKK2.dn) (30), pcDNA3-PK-I{kappa}B{alpha}S32A,S36A (31), and pSG5-Tax (32).

Purified anti-CD2 mAb (1 µg/ml) from the OX34 murine hybridoma (33) cross-linked with goat anti-mouse antibody (5 µg/ml; Sigma) was used to stimulate transfected 293 cells expressing rat CD2 chimeric proteins. Endogenous human p100/p52 was detected in Western blots using a commercial anti-p100 mAb (UBI 05-361). Anti-mouse p100N and anti-human p100N were used for detection of murine p100/p52 and immunoprecipitation of human p100/p52, respectively (20). Commercial antibodies purchased from Santa Cruz were used to detect the Myc epitope tag (sc-789), I{kappa}B{alpha} (sc-21), RelB (sc-226), and SAM68 (sc-333) on Western blots. Anti-LMP1-blotting antibody, CS.1-4 (34), was obtained from DAKO (M0897). CD2, EBNA2, EBNA LP, and LMP2A were detected in Western blots with OX34, PE2 (35), JF186 (36), and 14B7 (37) mAbs, respectively. Human anti-EBNA1 antiserum was kindly provided by Alan Rickinson (University of Birmingham, Birmingham, UK). Polyclonal antiserum to human T-cell lymphotrophic virus 1 (HTLV) Tax was obtained through the National Institutes of Health AIDS Research and Reference reagent program.

The proteasome inhibitor MG132 (Biomol Research Labs, 20 µM) and cycloheximide (Sigma; 10 µg/ml) were added 15-30 min before stimulation with anti-CD2 mAb and goat anti-mouse Ig antibody. Recombinant TNF{alpha} (20 ng/ml) was obtained from R & D Systems.

Cells—The Ramos tetracycline-regulated transactivator (tTA) and Ramos tTA-LMP1 cell lines have been described previously (38). Ramos tTA-LMP1 cells are stably transfected with two vectors; pJEF3 encoding a constitutively expressed tTA and pJEF6neo, in which LMP1 is cloned downstream of a promoter containing tTA binding sites. The Ramos tTA cell line is transfected only with pJEF3 vector. Ramos tTA-LMP1 and Ramos tTA cells were cultured in a complete medium comprising RPMI 1640 (Sigma) supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (50 units/ml), 300 µg/ml hygromycin B, and 1 µg/ml tetracycline (Roche Applied Science). For Ramos tTA-LMP1 cells, 1 mg/ml G418 (Invitrogen) was also included in the culture medium to ensure retention of pJEF6neo-LMP1 vector. To induce LMP1 expression, cells were extensively washed in phosphate-buffered saline and then recultured for the indicated times in complete medium without added tetracycline.

BL41 cells and their B95.8 EBV-infected derivative BL41+B95 cells were cultured as described previously (39). Mouse embryonic fibroblasts (MEFs) lacking IKK1 or IKK2 and control wild type MEFs were kindly provided by the originating laboratories (40, 41) and were cultured as described (42).

Protein Analyses—293 cells (5 x 105 cells/60-mm Nunc tissue culture dish) were transiently transfected using LipofectAMINE (Invitrogen). Amounts of plasmids used are indicated in the figure legends. Cells transfected with vectors encoding CD2-LMP 192 or tCD2 were stimulated as described in the figure legends. Whole cell lysates were prepared using buffer A (43), which was supplemented with 0.5% deoxycholate and 0.1% SDS (RIPA buffer). p100 ubiquitination experiments were performed as previously described (20). Equal protein loading of whole cell lysates was confirmed by Western blotting for tubulin.

Preparation of cytoplasmic and nuclear fractions was performed as described (44). Fractionation efficiency and protein loading were controlled by Western blotting for cytoplasmic (tubulin) and nuclear (SAM68) markers. To facilitate NF-{kappa}B subunit detection in Western blots, ~5-fold more cell equivalents of nuclear extract (10-25 µg) was loaded relative to cytoplasmic extract (30 µg). For immunoprecipitation experiments, nuclear and cytoplasmic extracts were diluted in buffer A (43) and adjusted to 10% (v/v) glycerol and 150 mM NaCl before incubation with anti-p100N antibody.

NF-{kappa}B Electrophoretic Mobility Shift Assays (EMSAs)—EMSAs and antibody supershifting were performed as described (44). A radiolabeled double-stranded oligonucleotide corresponding to the NF-{kappa}B binding site in the mouse immunoglobulin enhancer (Promega) was used to detect NF-{kappa}B complexes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LMP1 Induces NF-{kappa}B2 p52 Production in Transfected 293 Epithelial Cells—It has previously been reported that LMP1 expression in epithelial cells is associated with increases in p100 and p52 levels (45). However, it is unclear from this study whether other EBV-encoded latent proteins can also induce such changes. To address this question, LMP1, LMP2A, EBNA1, EBNA2, EBNA LP (Fig. 1A), and EBNA 3C (data not shown) were individually expressed in 293 epithelial cells. Western blotting of cell lysates indicated that none of the EBNA proteins or LMP2A had any effect on endogenous p100/p52 levels compared with empty vector (EV) control. In contrast, LMP1 expression stimulated a dramatic increase in steady state levels of p52 together with a more modest increase in p100. Therefore, of the panel of latent proteins tested, LMP1 was unique in its ability to increase p52 levels.



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  		FIG. 1.
LMP1 induces p52 production in transfected 293 epithelial cells. A, 293 cells were transfected with 500 ng of vector encoding LMP1 or 1 µg of vector encoding other EBV latent viral proteins or empty vector control (EV). After 48 h cells were lysed in RIPA buffer and Western-blotted for the indicated proteins. B, 293 cells were transfected with the indicated vectors (200 ng). After 42 h cells were incubated with anti-CD2 mAb ({alpha}CD2) cross-linked with goat anti-mouse Ig antibody (G{alpha}M) for the times shown and then lysed in RIPA buffer. Lysates were Western-blotted with the indicated antibodies.

 
Previous studies have indicated that signaling from the LMP1 cytoplasmic tail can be induced by cross-linking of chimeric molecules comprising tCD2 fused to the C-terminal 192 amino acids of LMP1 (CD2-LMP 192; Ref. 25). CD2-LMP 192, transiently expressed in 293 cells, induced an increase in endogenous p52 levels after 3-6 h of extensive cross-linking with anti-CD2 mAb, whereas p100 levels were largely unaffected (Fig. 1B). The time course of the p52 increase was similar to that previously observed with cross-linked CD40 (20) and was markedly slower than I{kappa}B{alpha} proteolysis induced by CD2-LMP 192, which was detectable at 30 min. (Fig. 1B). Cross-linked tCD2 failed to induce any alteration in p52 levels, whereas tCD2 fused to full-length wild type LMP1 (CD2-LMP1), which signals constitutively (25), increased p52 levels without anti-CD2 mAb cross-linking. These data suggest that stimulation of p52 levels by the LMP1 requires aggregation of its cytoplasmic tail.

LMP1 Induces p52 as a Consequence of Proteasome-mediated p100 Proteolysis—To determine whether NF-{kappa}B-dependent gene expression is required for LMP1-induced p52 production, 293 cells were co-transfected with LMP1 vector together with an expression vector encoding a super-repressor mutant of I{kappa}B{alpha} (I{kappa}B{alpha}SS/AA), which blocks NF-{kappa}B activation (31), or EV. Expression of I{kappa}B{alpha}SS/AA on its own reduced the basal levels of endogenous p100 expression compared with EV alone and blocked the ability of LMP1 to increase the steady state levels of p100. However, LMP1 still clearly induced p52 levels when co-expressed with I{kappa}B{alpha}SS/AA while concomitantly reducing levels of p100 (Fig. 2A). Thus, LMP1 induces p52 production independently of NF-{kappa}B activation.



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  		FIG. 2.
LMP1-mediated p52 production requires proteasome activity and de novo protein synthesis but is independent of NF-{kappa}B activity. A, 293 cells were transfected with 200 ng of vectors encoding LMP1 or EV together with 800 ng of plasmid encoding I{kappa}B{alpha}SS/AA or EV. After 48 h cells were lysed in RIPA buffer and Western-blotted. I{kappa}B{alpha}SS/AA was expressed at a similar level in all transfections as determined by Western blot analysis (data not shown). B, 293 cells were transfected with CD2-LMP 192 vector (200 ng). Cells were cultured for 42 h, preincubated for 20 min with MG132, cycloheximide (CHX), or vehicle control, and then stimulated for the indicated times with cross-linked anti-CD2 mAb ({alpha}CD2/G{alpha}M). Western blots of RIPA cell lysates were probed as shown. C, 293 cells were co-transfected as indicated with 500 ng of vector encoding Myc-p100 or EV control (-) together with 1000 ng of plasmid encoding HA-ubiquitin (HA-Ubi) or EV (-) and 500 ng of NIK or LMP1 plasmid or EV control. After 24 h culture, cells were incubated for 3 h with MG132 (50 µM) and lysed in buffer A, and p100 was immunoprecipitated. Immunoprecipitates (Ip) were resolved on a 6% SDS-polyacrylamide gel and Western-blotted. D, 293 cells were co-transfected with 200 ng of plasmid encoding LMP1 or EV together with 200 ng of plasmids encoding Myc-p100 (WT), Myc-p100{Delta}GRR ({Delta}GRR), or Myc-p100S866A,S870A (SS/AA). After 48 h cells were lysed in RIPA buffer and Western-blotted. NS, non-specific.

 
To investigate the role of the proteasome in LMP1-mediated p52 production, 293 cells were transfected with a vector encoding CD2-LMP 192 to facilitate analysis of the acute up-regulation of p52 by LMP1. Increases in p52 induced by cross-linked CD2-LMP 192 were blocked by pretreatment of cells with the proteasome inhibitors MG132 (Fig. 2B) or N-acetyl-leu-leu-norleucinal (data not shown), demonstrating a requirement for proteasome activity for LMP1 induction of p52.

Protein ubiquitination is often a prerequisite for proteasome action. Therefore, experiments were conducted to determine whether LMP1 induced the ubiquitination of p100. 293 cells were co-transfected with plasmids encoding Myc-p100 and HA-ubiquitin together with LMP1 or EV. LMP1-induced polyubiquitination of Myc-p100 was clearly demonstrated by the appearance of high molecular weight bands in Western blots of immunoprecipitated p100 probed for HA-ubiquitin (Fig. 2C). NIK expression also induced Myc-p100 ubiquitination (Fig. 2C), as reported previously (22). Thus, proteasome-mediated production of p52 induced by LMP1 involves p100 ubiquitination. Furthermore, the observation that LMP induces p52 levels concomitantly with decreases in p100 levels when NF-{kappa}B activity is blocked (Fig. 2A) strongly suggests that LMP1 induces p52 production via proteasome-mediated proteolysis of p100.

Two motifs in p100 have been previously shown to be required for CD40-induced processing of p100 to p52 (20); namely the glycine-rich region (GRR; residues 346-377) (46) and two serines (Ser-866 and Ser-870) in the C-terminal PEST region of p100 that are thought be phosphorylated by IKK1 (21). To investigate whether the GRR and serines 866/870 are important in LMP1-triggered p52 production, 293 cells were co-transfected with plasmids encoding LMP1 or EV and either wild type Myc-p100, Myc-p100{Delta}GRR, or Myc-p100S866A,S870A. LMP1 co-transfection with wild type Myc-p100 significantly increased Myc-p52 levels compared with EV control (Fig. 2D). However, LMP1 failed to induce Myc-p52 from either Myc-p100{Delta}GRR or Myc-p100S866A,S870A (Fig. 2D). Therefore, both the GRR and serines 866 and 870 in the PEST region of p100 are required for LMP1-induced p52 production from p100. These results further support the conclusion that LMP1 induces p52 as a consequence of p100 processing.

LMP1 Induction of p52 Requires Protein Synthesis—Both CD40 (20) and B cell-activating factor receptor (17) have a requirement for de novo protein synthesis to trigger p100 processing. The requirement for protein synthesis in CD2-LMP1 192-induced p52 production, which occurs over several hours (Fig. 1B), was therefore investigated. 293 cells were transfected with an expression vector encoding CD2-LMP 192 and cycloheximide was added 30 min before CD2-LMP 192 cross-linking with anti-CD2 mAb. Cycloheximide treatment inhibited p52 production induced by cross-linked CD2-LMP 192 (Fig. 2B). Cycloheximide also blocked CD2-LMP 192 induction of p52 when added simultaneously with anti-CD2 mAb but not when added 2 h after CD2 cross-linking (data not shown). These results, which show a striking similarity to those reported for CD40 (20), demonstrate that de novo protein synthesis is required during the first 2 h of cross-linked CD2-LMP 192 signaling to induce p100 processing efficiently.

LMP1 Induces the Nuclear Translocation of p52 and RelB in 293 Epithelial Cells—p100 is the major I{kappa}B for RelB in the cell (47). Therefore, one predicted outcome of LMP1-mediated p100 processing is the nuclear translocation of p52·RelB complexes. To initially investigate this, cytoplasmic/nuclear fractions were prepared from 293 cells transfected with expression vectors encoding CD2-LMP 192 or tCD2, stimulated with anti-CD2 mAb, or left unstimulated. Efficient separation of the cytoplasmic and nuclear fractions was demonstrated for this (Fig. 3A) and subsequent experiments by Western blotting for cytoplasmic (tubulin) and nuclear (SAM68) markers. In unstimulated or control tCD2-transfected cells, very little endogenous p52 or RelB was detected in the nuclear fraction (Fig 3A). Cross-linking of CD2-LMP 192 for 30 min resulted in a small increase in nuclear p52 but had no effect on nuclear RelB levels. This small increase in nuclear p52 at 30 min is likely to be due to the release of pre-existing p52 bound to small I{kappa}Bs, including I{kappa}B{alpha} (Fig. 1B; Ref. 20). However, marked increases in both nuclear p52 and RelB were detected after 6 h of cross-linking of CD2-LMP 192, although cytoplasmic RelB levels were unaffected. Moreover, immunoprecipitation of nuclear extracts with anti-p100N antibody demonstrated that the translocated nuclear p52 and RelB are associated with one another (Fig. 3B). These experiments indicate that cross-linked CD2-LMP 192 stimulates p100 processing, resulting in the release of RelB, which translocates to the nucleus in a complex with p52.



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  		FIG. 3.
LMP1-induces the nuclear translocation of p52 and RelB in 293 epithelial cells.A, 293 cells were transfected with vectors encoding CD2-LMP 192 (600 ng) or tCD2 control vector (700 ng). Different amounts of DNA were used to normalize protein expression levels. After 42 h cells were stimulated with cross-linking antibody for the times indicated. Cytoplasmic and nuclear cell fractions were prepared and Western-blotted. B, 293 cells were transfected with vector encoding CD2-LMP 192 (200 ng). After 42h cells were stimulated with cross-linking antibody for the times indicated, and cytoplasmic and nuclear cell fractions were prepared. Control (IgG) and p52 immunoprecipitations (Ip) were carried out on nuclear extracts and Western-blotted as indicated. C, 293 cells were transfected with vectors encoding LMP1 (200 ng) or EV (200 ng). After 48 h cytoplasmic and nuclear fractions were prepared and Western-blotted. D, 293 cells were transfected with vectors encoding LMP1 (200 ng) or EV (200 ng). After 48 h cytoplasmic and nuclear fractions were prepared. Control (IgG) and anti-p100N immunoprecipitations were carried out on cytoplasmic extracts. Cytoplasmic extracts pre- and post-immunoprecipitation were analyzed by Western blotting as indicated.

 
Importantly, expression of wild type LMP1 in 293 cells also resulted in nuclear translocation of both p52 and RelB (Fig. 3C). Unlike the CD2-LMP192 construct, wild type LMP1 caused a concomitant increase in the steady state levels of cytoplasmic RelB (Fig. 3C). However, p100 depletion experiments using a p100 C-terminal directed antiserum indicated that essentially all the cytoplasmic RelB was associated with p100 in LMP1-transfected 293 cells (Fig. 3D). This strongly suggests that LMP1 promotes RelB nuclear translocation as a result of p100 processing rather than simply through an increase in steady state levels of RelB protein.

EMSAs demonstrated that LMP1 expression induced marked activation of NF-{kappa}B binding activity, which was of a similar magnitude to that detected in cells stimulated for 20 min with TNF{alpha} (Fig. 4A). Supershift EMSA analyses indicated that the majority of DNA binding activity induced by LMP1 comprised p50 and p65 NF-{kappa}B subunits (Fig. 4B, left panel). However, a p52-containing NF-{kappa}B complex was also clearly detectable in LMP1-transfected cells (Fig. 4B, right panel). Thus, LMP1-induced nuclear p52 can bind to specific {kappa}B sites in target DNA.



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  		FIG. 4.
LMP1 induces NF-{kappa}B DNA binding activity that includes p52-containing complexes. A, NF-{kappa}B EMSAs were carried out on nuclear fractions generated from 293 cells transfected with EV (200 ng) or LMP1 vector (200 ng) or untransfected cells that were stimulated for 20 min with TNF{alpha} or left unstimulated. Specificity of {kappa}B binding was confirmed by competition with excess unlabeled control Oct-1 oligonucleotide (Oligo, Oct-1) or {kappa}B oligonucleotide (Oligo, {kappa}B). B, nuclear extracts from 293 cells transfected with LMP1 vector (200 ng) were supershifted with the indicated antibodies to different NF-{kappa}B subunits or control antibody (IgG). The position of a p52-containing supershifted NF-{kappa}B complex is shown (asterisk).

 
LMP1 Induces Nuclear Translocation of p52 and RelB in Ramos B Cells—Although EBV can infect epithelial cells, the major cell type targeted in EBV infection is the B cell (3). Furthermore, LMP1 plays an essential role in maintaining the transformed phenotype of EBV-infected B lymphoblastoid cells (48). It was, therefore, important to determine whether LMP1 could induce p100 processing to p52 in B cells as well as epithelial cells. To address this question, an EBV-negative Ramos B cell line was used that had been stably transfected with a vector system which allows LMP1 expression to be inducibly regulated by tetracycline (Tet) (26). In this system, when Tet is removed from growth medium, the constitutively expressed tTA can bind to sites in the vector promoter, resulting in LMP1 gene transcription.

Removal of Tet from the growth medium resulted in a clear increase in steady state levels of LMP1 protein in whole cell extracts from tTA-LMP1-transfected Ramos cells, which paralleled a dramatic increase in p52 levels (Fig. 5A). In contrast, no change in p52 levels was detected in control Ramos tTA cells upon removal of Tet. LMP1 expression also resulted in increased steady state levels of p100 in addition to increased p52 levels (Fig. 5A). Because transcription of p100 is an NF-{kappa}B-regulated event (49), increases in p100 presumably arise due to LMP1-induced NF-{kappa}B activation. Even in the presence of Tet, Ramos tTA-LMP1-transfected cells contained increased steady state levels of p100 and p52 compared with control Ramos tTA-transfected cells (Fig. 5A). This is probably due to low levels of LMP1 transcription in the absence of promoter-bound tTA.



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  		FIG. 5.
LMP1 induces p52 production and nuclear translocation of p52/RelB dimers in Ramos B cells. tTA and tTA/LMP1 stable Ramos B cells were washed free of tetracycline and re-cultured in tetracycline-free medium for the times indicated. A, cells were lysed in RIPA buffer and Western-blotted. B, cytoplasmic and nuclear fractions were prepared and Western-blotted. C, NF-{kappa}B EMSAs were carried out on nuclear fractions either alone or in conjunction with p52 supershift analysis, as indicated. The position of a p52-containing supershifted NF-{kappa}B complex is shown (asterisk). D, control (IgG) and p52 immunoprecipitations were carried out on nuclear extracts. Immunoprecipitates (Ip) were Western-blotted for p52 and RelB, as indicated.

 
To determine whether increased levels of p52 induced by LMP1 expression contribute to NF-{kappa}B activation, nuclear extracts were prepared from Ramos tTA-LMP1 B cells in the presence and absence of Tet. Nuclear p52 was barely detectable by Western blot analysis in the presence of Tet. However, upon LMP1 induction, nuclear p52 levels markedly increased (Fig. 5B). In control tTA cells, nuclear p52 was not detectable in the presence or absence of Tet (Fig. 5B). Supershift EMSAs confirmed that LMP1-induced p52 in Ramos tTA LMP1 cells was able to bind to target DNA containing an NF-{kappa}B binding site (Fig. 5C). These p52-containing complexes only represented a small fraction of NF{kappa}B binding activity induced, which was predominantly composed of p50 and RelA (data not shown).

In Ramos B cells, as in other cell types (47), p100 is the only cytoplasmic I{kappa}B that binds to RelB, as determined by co-immunoprecipitation experiments (data not shown). Therefore, any nuclear translocation of RelB upon NF-{kappa}B activation is likely to arise from p100 processing. Blotting nuclear extracts from Ramos tTA-LMP cells revealed that RelB levels increased in parallel with p52 after LMP1 induction (Fig. 5B). Furthermore, immunoprecipitation of nuclear extracts with anti-p100N antibody clearly showed that nuclear RelB is associated with p52 (Fig. 5D). These data suggest that LMP1 expression induces p100 processing in Ramos B cells.

EBV Infection Results in Nuclear Translocation of p52-RelB Heterodimers—It was important to determine whether EBV infection of B cells, which involves expression of LMP1 (23), also induces p100 processing to p52. To investigate this question, cell extracts were prepared from BL41 Burkitt lymphoma cells and their B95/8 EBV-infected derivative, BL41+B95 (39). Western blotting demonstrated a clear increase in both p100 and p52 after EBV infection (Fig. 6A). Subcellular fractionation of the two cell lines revealed a dramatic increase in nuclear p52 induced by EBV infection (Fig. 6B). Immunoprecipitation of nuclear lysates with anti-p100N antibody confirmed that nuclear p52 was associated with RelB (Fig. 6C). These data suggest that the alternative NF-{kappa}B-signaling pathway which triggers p100 processing is activated as a consequence of EBV infection of B cells.



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  		FIG. 6.
EBV infection results in 52 production and nuclear translocation of p52/RelB dimers in BL41 + B95 B cells. A, EBV negative (-) and EBV positive (+) BL41 cells were lysed in RIPA buffer and Western-blotted. B, cytoplasmic and nuclear fractions were prepared from EBV negative (-) and EBV positive (+) BL41 cells and Western-blotted. C, control (IgG) and p52 immunoprecipitations were carried out on nuclear extracts from EBV negative (-) and EBV positive (+) BL41 cells. Immunoprecipitates (Ip) were Western-blotted for p52 and RelB, as indicated.

 
CTAR1 Is Required for LMP1 Stimulation of p100 Processing—Previous research has indicated that optimal induction of "global" NF-{kappa}B activity by LMP1 requires both CTAR1 and CTAR2, with CTAR2 having a dominant role (50-52). However, it is unclear to what extent CTAR1 and CTAR2 contribute to the activation of the canonical and alternative NF-{kappa}B pathways, which together generate the total NF-{kappa}B activity detected in EMSAs. To address this question, 293 epithelial cells were transfected with vectors encoding wild type or mutant versions of LMP1 with defective CTAR1 and/or CTAR2 signaling (Fig. 7, A-C). LMPAAA contains inactivating point mutations in CTAR1, LMPY384G contains an inactivating point mutation in CTAR2, and LMPAAA/Y384G is defective in signaling from both CTAR1 and CTAR2 (53, 54).



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  		FIG. 7.
Differential requirements for CTAR1 and 2 in LMP1-induced NF-{kappa}B activation. A and B, 293 cells were transfected for 48 h with the indicated amounts of vectors, which were adjusted to normalize LMP1 protein expression: LMP1 WT (300 ng), CTAR1 mutant LMPAAA (AAA; 500 ng), of CTAR2 mutant LMPY384G (Y384G; 400 ng), LMP1 double mutant LMPAAA/Y384G (AAA/Y384G; 400 ng), or EV control (500 ng). A, 45-h post-transfection cells were preincubated with 20 µM MG132 for 2 h and, where indicated, further stimulated with TNF{alpha} for 30 min. Cell lysates were immunoprecipitated with anti-I{kappa}B{alpha} antibody. Immunoprecipitates and lysates were Western-blotted as shown. B, transfected cells were lysed in RIPA buffer and Western-blotted. C, cytoplasmic and nuclear fractions were prepared 48 h after transfection of 293 cells with plasmids encoding LMP1 WT (200 ng), LMP1 AAA (300 ng), LMP1 Y384G (200 ng), or EV control (300 ng) and Western-blotted as indicated.

 
A Western blot assay for phosphorylation of I{kappa}B{alpha} on serine 32, which is directly phosphorylated by the IKK complex (15), was used as a readout for activation of the canonical NF-{kappa}B pathway. Wild type LMP1 expression resulted in robust I{kappa}B{alpha} phosphorylation of similar magnitude to that observed after 15 min of TNF{alpha} stimulation of EV-transfected cells (Fig. 7A). In contrast, expression of LMPY384G or LMPAAA/Y384G induced little detectable I{kappa}B{alpha} phosphorylation. LMPAAA was able to elicit marked I{kappa}B{alpha} phosphorylation, although not as great as with wild type LMP1 (Fig. 7A). Thus, activation of the canonical NF-{kappa}B pathway, which triggers I{kappa}B{alpha} phosphorylation, is primarily mediated via CTAR2.

The effect of each of the LMP1 mutants was also tested on activation of the alternative NF-{kappa}B pathway leading to p52 production. As noted previously (Figs. 1 and 2), wild type LMP1 expression increased levels of both p100 and p52 compared with EV (Fig. 7B). However, although expression of the LMPAAA increased p100 levels similar to wild type LMP1, this mutant had no effect on p52 levels. Conversely, LMPY384G expression induced steady state levels of p52 to a similar degree to wild type LMP1 but had little effect on p100 levels (Fig. 7, B and C). No increases in p100 or 52 were detected with expression of LMPAAA/Y384G (Fig. 7B). Consistent with these results, expression of wild type LMP1 or LMPY384G but not LMPAAA or LMPAAA/Y384 induced the nuclear translocation of p52 and RelB (Fig. 7C). In addition, LMPAAA, which can efficiently activate the canonical NF-{kappa}B pathway (Fig. 7A), induced increases in the cytoplasmic levels of p100 and RelB without coordinately stimulating their nuclear translocation (Fig. 7C). This suggests that transcriptional activation of the NF-{kappa}B-sensitive genes RelB and p100 (47, 55), requires signals from CTAR2, which activates the canonical NF-{kappa}B pathway. Maximal activation of p100 processing, which results in nuclear translocation of p52 and RelB, in contrast requires signals from CTAR1.

LMP1-mediated p100 Processing Is Dependent on NIK and IKK1 but Independent of IKK2—The mitogen-activated protein 3-kinase NIK is required for p100 processing induced by LT{beta}-R, B cell-activating factor receptor, and CD40 (17, 19, 20) but not by the HTLV-transforming protein, Tax (56). To investigate whether LMP1-induced processing of p100 requires NIK, 293 epithelial cells were co-transfected with expression vectors encoding wild type LMP1 or Tax and the C-terminal portion of NIK (NIK{Delta}N; residues 624-947), which functions as a potent inhibitor of wild type NIK (57), or EV. LMP1-mediated increases in p52 but not p100 were clearly inhibited with the co-expression of NIK{Delta}N (Fig. 8A). In contrast, NIK{Delta}N expression had little effect upon Tax-induced p100 processing, indicating that NIK{Delta}N was acting in a specific manner (Fig. 8A). These results suggest that LMP1 induces p100 processing via a NIK-dependent pathway.



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  		FIG. 8.
LMP1-mediated p100 processing is dependent upon NIK and IKK1 but independent of IKK2. A, 293 cells were co-transfected with plasmids encoding Myc-NIK{Delta}N (1 µg) or EV control (1 µg) and Tax (600 ng) or LMP1 (400 ng). All transfections contained 1.6 µg of DNA, made up with EV where appropriate. After 48 h cells were lysed in RIPA buffer and Western-blotted. B, 293 cells were transfected with the indicated quantities of plasmid to achieve similar protein levels: EV alone (1.5 µg), EV and IKK1.dn (0.5 and 1 µg, respectively), EV and IKK2.dn (1.3 and 0.2 µg), LMP1 and EV (0.1 and 1.4 µg), LMP1 and IKK1.dn (0.2 and 1 µg), and LMP1 and IKK2.dn (0.2 and 0.2 µg). Total DNA transfected per dish was 1.5 µg, made up with EV where appropriate. After 48 h cells were lysed in RIPA buffer, and extracts were Western-blotted. C, plasmids encoding LMP1 or empty vector (-) were transfected into MEFs. Amounts of plasmid DNA used were adjusted to normalize LMP1 expression levels: WT MEF (500 ng), IKK1-/- (750 ng), and IKK2-/- (3000 ng). Cytoplasmic and nuclear extracts were prepared and Western-blotted for the indicated proteins.

 
To initially investigate the role of IKK1 and IKK2 in LMP1-induced p100 processing, the effect of dominant negative (dn) versions of each of these kinases was investigated in 293 cells. These both contain two serine to alanine mutations in the activation loops of their kinase domains (30). IKK1.dn markedly reduced the level of p52 detected in LMP1 co-expressing cells compared with EV (Fig. 8B). However, IKK1.dn had little effect on the LMP1-mediated increase in p100. Conversely, expression of IKK2.dn blocked the LMP1-mediated increase of p100 levels but had little effect on p52 up-regulation (Fig. 8B). These data suggest that IKK1 is required for LMP1 triggering of the alternative NF-{kappa}B pathway regulating p100 processing to p52, whereas IKK2 is required for transcriptional up-regulation of p100.

The role of IKK1 and IKK2 was further investigated using knockout MEFs lacking individually IKK1 (40) or IKK2 (41). LMP1 was transiently expressed in WT, IKK1-/- and IKK2-/- MEFs, and p100 processing was monitored by assaying the nuclear translocation of p52 and RelB. LMP1 expression increased cytoplasmic p100 and RelB levels in both WT and IKK1-/- MEFs but only marginally in IKK2-/- MEFs (Fig. 8C). Furthermore, LMP1 expression in WT and IKK2-/- resulted in clear nuclear translocation of p52 and RelB (Fig. 8C). However, in IKK1-/- MEFs, LMP1 expression failed to induce nuclear translocation of either of these Rel subunits. LMP1 expression was also shown to stimulate the p38 mitogen-activated protein kinase phosphorylation to a similar degree in each of the MEFs (Fig. 8C), confirming that the transfection efficiency of LMP1 was similar in each of the transfected cell lines. Thus, there is a specific requirement for IKK1 in LMP1-induced p100 processing and for IKK2 in LMP1-induced p100 and RelB up-regulation.

Taken together these results demonstrate that LMP1-induced p100 processing requires NIK and IKK1 but not IKK2. However, LMP1 induced up-regulation of the p100 and RelB, which are both NF-{kappa}B-sensitive genes (47, 55), requires IKK2 but not IKK1. These data are consistent with recent published studies on LT{beta}-R signaling (19), in which it has been shown that NIK and IKK1 are required for p100 processing to p52 induced by LT{beta}-R, whereas IKK2 is required for LT{beta}-R up-regulation p100 expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies using an I{kappa}B{alpha} super-repressor mutant indicate that NF-{kappa}B activation is critical for transformation of fibroblasts by LMP1 (12) and for survival of EBV-transformed LCLs (11). Although these experiments have firmly established an important role for NF-{kappa}B in cell transformation by EBV, the potential role of the alternative NF-{kappa}B pathway in EBV-mediated cell transformation is not known. In the present study, LMP1 is shown to stimulate p100 processing to p52. This results in the nuclear translocation of p52-RelB heterodimers in epithelial cells and B cells, which are two cell types targeted by EBV infection. Thus, LMP1 simultaneously triggers both canonical and alternative NF-{kappa}B pathways. Interestingly, Tax, the transforming protein of HTLV type I, also activates both NF-{kappa}B pathways (56, 58, 59). The simultaneous activation of canonical and alternative NF-{kappa}B pathways, therefore, may facilitate viral propagation.

The activation of p100 processing by LMP1 extends the number of signaling pathways that it activates in common with CD40 (20, 23). Indeed, it is likely that both LMP1 and CD40 trigger p100 processing via a similar mechanism. Thus, a TRAF binding site in the cytoplasmic tail of both proteins is required to induce p52, and both proteins require NIK activity to trigger this pathway (Figs. 7B and 8A; Ref 20). The present study further demonstrates that LMP1-mediated processing is a delayed event (Fig. 1B) that requires de novo protein synthesis but is independent of NF-{kappa}B activation (Fig. 2, A and B), similar to CD40 (20).

Supershift EMSAs indicate that p52 containing NF-{kappa}B dimers make up only a minor component of total NF-{kappa}B DNA binding activity induced by either LMP1 or CD40 (Figs. 4B and 5C; Ref. 20). Nevertheless, analyses of NF-{kappa}B2 (60, 61) and RelB (62, 63) knockout mice reveal that p52 and RelB perform functions that cannot be compensated by other Rel subunits. Therefore, activation of the alternative NF-{kappa}B pathway by endogenous cell surface receptors is likely to be important for their functions (16). Consistent with this hypothesis, p100 processing has been implicated in B cell-activating factor receptor-mediated B cell development and survival (17, 18). Moreover, two subsets of NF-{kappa}B-regulated genes induced by LT{beta}-R have been described, one activated by the canonical pathway and the other by the alternative pathway (19). Notably, both RelB (64) and NF-{kappa}B2 (60) have been shown to be required for optimal CD40-induced B cell proliferation and are therefore, by analogy, most likely required for the proliferative signals generated by LMP1. Thus, activation of the alternative NF-{kappa}B pathway by LMP1 is likely to play an important role in its transforming function.

CTAR1 and CTAR2 have distinct functions in the activation of NF-{kappa}B by LMP1. Thus, CTAR1 has only a minor role in activating the canonical NF-{kappa}B pathway that controls I{kappa}B{alpha} phosphorylation (Fig. 7A) but is essential for maximal stimulation of p100 processing to p52 (Fig. 7B). In contrast, the majority of LMP1-triggered I{kappa}B{alpha} phosphorylation requires CTAR2 function, but this is dispensable for induction of p100 processing (Fig. 7, A and B). CTAR2 function is also required for LMP1-mediated increases in total protein levels of p100 and RelB. This suggests that activation of the canonical NF-{kappa}B pathway is responsible for transcriptional up-regulation of these genes but is not able to induce nuclear translocation of p52 and RelB, consistent with earlier studies on LT{beta}-R signaling (19). The two IKK subunits also have distinct functions in LMP-1 induction of NF-{kappa}B. Thus, IKK2 is required for up-regulation of p100 and RelB via the canonical NF-{kappa}B pathway, whereas IKK1 regulates the alternative pathway that triggers p52 production and p52/RelB nuclear translocation (Fig. 8, B and C). This implies that LMP1 CTAR1 predominantly triggers the IKK1-dependent alternative NF-{kappa}B pathway, whereas LMP1 CTAR2 triggers the IKK2-dependent canonical NF-{kappa}B pathway.

The critical role of NF-{kappa}B activation for EBV- or LMP1-mediated cell transformation is highlighted by studies showing that global inhibition of NF-{kappa}B activity triggers apoptosis of EBV-transformed LCLs (11) and blocks LMP1 transformation of Rat1 fibroblasts (12). However, the relative importance of the canonical and alternative pathways of NF-{kappa}B activation in EBV- and LMP1-induced transformation is not known. Nevertheless, it is intriguing that in EBV-infected B cells, CTAR1 is required for the initial establishment of the transformed phenotype, whereas CTAR2 signals maintain long term cell proliferation (4, 65-67). It is possible, therefore, that the persistent activation by LMP1 CTAR1 of the alternative NF-{kappa}B pathway contributes to the transforming potential of this protein. Consistent with this hypothesis, p52 is generated constitutively in many human lymphomas due to mutation of the nfkb2 locus by chromosomal translocations or deletions (68), and such lymphoma-associated NF-{kappa}B2 proteins can transform murine fibroblasts (69). The possible role of p100 processing in cell transformation by LMP1 is currently being investigated.


   FOOTNOTES
 
* This work was supported by the United Kingdom Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 44-8816-2463; Fax: 44-8816-2722; E-mail: sley{at}nimr.mrc.ac.uk.

1 The abbreviations used are: EBV, Epstein-Barr virus; IKK, I{kappa}B kinase; LMP, latent membrane protein; EBNA, EBV nuclear antigen; LP, leader protein; GRR, glycine-rich region; dn, dominant negative; LCL, lymphoblastoid cell line; CTAR, C-terminal-activating region; TNF, tumor necrosis factor; LT{beta}-R, lymphotoxin {beta} receptor; HA, hemagglutinin; NIK, NF-{kappa}B inducing kinase; mAb, monoclonal antibody; tTA, tetracycline-regulated transactivator; MEF, mouse embryonic fibroblast; RIPA, radioimmune precipitation assay buffer; EMSA, electrophoretic mobility shift assay; EV, empty vector; Tet, tetracycline; HTLV, human T-cell lymphotrophic virus. Back


   ACKNOWLEDGMENTS
 
We thank Shizuo Akira, Dirk Bohmann, Gilles Courtois, Ron Hay, Alain Israel, Mike Karin, Arnd Kieser, Gary Nabel, Hiroyasu Nakano, and Alan Rickinson for reagents used in this study. Members of the Ley laboratory are also gratefully acknowledged for help during this study.



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