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The Journal of Immunology, 1999, 163: 6261-6268.
Copyright © 1999 by The American Association of Immunologists

Inactivation of NF-{kappa}B by EBV BZLF-1-Encoded ZEBRA Protein in Human T Cells1

David H. Dreyfus, Masayuki Nagasawa, Joanne C. Pratt, Colm A. Kelleher and Erwin W. Gelfand2

Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that the EBV ZEBRA protein (also denoted EB1, Z, or Zta) encoded by the BZLF open reading frame is expressed in primary human thymocytes and in human T lymphoblastoid cell lines infected by EBV. Expression of EBV-encoded gene products in T lymphocytes could contribute to viral pathogenesis during acute EBV infection as well as in individuals coinfected with EBV and HIV. HPB-ALL and Jurkat T lymphoblastoid cell lines transiently and stably expressing ZEBRA were characterized in this work. Expression of ZEBRA protein in human T lymphoblastoid cells was associated with decreased expression of an NF-{kappa}B reporter gene, altered expression of the NF-{kappa}B p50 protein subunit, and decreased DNA binding by components of NF-{kappa}B. These observations suggest that inactivation of NF-{kappa}B transcription by ZEBRA in EBV-infected T cells may be a novel mechanism of viral pathogenesis analogous in part to over-expression of the endogenous cytoplasmic inhibitor of NF-{kappa}B, I{kappa}B{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epstein-Barr virus infects a variety of human cell types including B and T lymphocytes and epithelial cells, although stable latency is established only in B lymphocytes (1). During acute infectious mononucleosis, EBV-infected T lymphocytes are evident (2, 3), and the EBV genome is often detected in T cell tumors (4, 5). EBV infection of T lymphocytes may also play a role in the pathogenesis of the hemophagocytic syndrome (6). T lymphocytes obtained from patients with active EBV infection exhibit an abnormal morphology (7), increased apoptosis (8, 9) and altered signaling through the TCR (10). EBV infection could alter T cell function indirectly by the effects of a viral-encoded superantigen (11), or more directly by expression of EBV-encoded gene products in T lymphocytes (12, 13, 14, 15, 16, 17). Because T lymphocytes are required for suppression of EBV replication (2), a loss of functional T cells could lead to increased viral replication in EBV-infected tissues during acute viral infection. However, the pathogenesis of EBV is less well characterized in T lymphocytes than in either B lymphocytes or epithelial cells, and the significance of T cell infection remains to be established in immunocompetent individuals.

The means through which EBV infects T lymphocytes in vivo may differ at different stages of cellular maturation. Both HPB-ALL (18) and Jurkat T lymphoblastoid cells (19) express a molecule similar to B lymphocyte CD21 and can be infected with EBV, although other CD21-like molecules expressed on T lymphocytes may also bind and internalize EBV (reviewed in Ref. 20). Several model systems have been used to characterize EBV infection of T cells. In one model, infection of primary thymocytes in vitro (12, 13, 14, 15), EBV appeared to target a population of immature CD4+/CD8+ thymocytes that express high levels of the CD21 complement receptor (the major EBV receptor on B lymphocytes). In this system, infection of cells seemed to correlate with levels of CD21 expression (13). In another model, infection of mature peripheral T cells in vitro did not appear to require expression of CD21 (16).

Expression of EBV gene products in T cells is also associated with increased replication of HIV in coinfected cells (16). Expression of genes typical of EBV type II latency (1) is evident in T cell lymphoma (21) and T lymphoblastoid cell lines infected under selective conditions with EBV (22). LMP-1 and EBNA-2 gene products expressed during viral latency have been transiently expressed in human T lymphoblastoid cells including Jurkat and HPB-ALL and shown to alter expression of T cell-surface markers including CD23 and ICAM (23). Stable expression of the LMP-1 gene product in Jurkat T lymphoblastoid cells reduces cellular apoptosis (24). In primary peripheral T lymphocytes infected with EBV in vitro (16), and in some EBV-infected T cells in vitro (3), the EBER transcripts typical of EBV latency are detected. The BRLF-1 transcript characteristic of viral reactivation is also expressed in primary peripheral T cells infected in vitro with EBV (16). Despite the expression of latency associated genes in T cell tumors and peripheral T cells, the inability of EBV to immortalize T cells under normal conditions of infection is a critical difference between EBV infection of T and B cells (17).

We have previously demonstrated that genes typical of EBV lytic growth are expressed in human thymocytes infected with EBV, although infectious virus is not produced at detectable levels (14). The BZLF-1-encoded ZEBRA protein that activates the viral lytic cycle is expressed in primary thymocytes infected by EBV, although not in most T cell tumors (21). Activation of the BZLF-1 promoter has also been characterized in the EBV-negative Jurkat T lymphoblastoid cell line, and expression of BZLF-1 requires activation of cAMP-dependent signaling (25). ZEBRA is a viral homologue of the cellular c-Fos transcription factor that binds to AP-1-like sequences termed ZEBRA response elements or ZRE3 (26, 27, 28). Unlike c-Fos, ZEBRA can also bind directly to the p65 (RelA) subunit of NF-{kappa}B (29, 30) and other cellular transcription factors including the retinoic acid receptors RAR and RXR (31), the TATA binding protein TFIID (32), the C/EBP transcription factor (33), and tumor suppressor p53 (34). Previous studies have demonstrated that transcriptional activity by ZEBRA is blocked in T-lymphoblastoid cells, apparently due to dimerization of ZEBRA with endogenous cytoplasmic transcription factors (29, 30, 31, 32).

The ability of ZEBRA to bind to cellular transcription factors such as NF-{kappa}B p65 suggested that complexes between ZEBRA and endogenous transcription factors could alter the function of these transcription factors in T cells. To test this hypothesis, we generated Jurkat T cell lines stably expressing ZEBRA protein and characterized the effects of ZEBRA expression upon NF-{kappa}B expression and function. These experiments suggest that binding between ZEBRA and the p65 (RelA) subunit of NF-{kappa}B may be functionally similar to binding of endogenous inhibitor of NF-{kappa}B, I{kappa}B{alpha} (35, 36). Inducible degradation of I{kappa}B{alpha} leads to activation of NF-{kappa}B transcription sites (37) and plays a central role in the regulation of inflammation (38), regulation of apoptosis (39, 40), and cytokine expression (41). Thus, transient expression of ZEBRA protein in T cells responding to EBV infection may selectively target and kill activated cells through inactivation of NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids

pSV2-neo-WZhet-expressing ZEBRA (42) and control plasmid pSV2-neo were obtained from Dr. G. Miller (Yale University, New Haven, CT). Luciferase reporter genes containing response elements for AP-1 (43) and NF-{kappa}B (37) fused to a minimal fos promoter element denoted pf-luc were obtained from Dr. G. Johnson (National Jewish Medical and Research Center, Denver, CO). Reporter plasmids were originally constructed and obtained from Drs. R. Flavell and S. Ghosh (Yale University). A plasmid expressing constitutive mitogen-activated kinase/extracellular signal-related kinase kinase-1 (MEKK1) denoted MEKK{Delta} (44) was also obtained from Dr. G. Johnson. A plasmid expressing constitutively active I{kappa}B{alpha} (45) denoted {Delta}N1 (I{kappa}B{alpha} with deletion of amino acids 1–36) was obtained from Dr. L. Ghoda (University of Colorado Health Sciences Center, Denver, CO). Plasmids were prepared using either Promega (Madison, WI) midi-preps or Qiagen (Valencia, CA) systems.

Cells and cell culture

HPB-ALL cells were as described previously (14). Jurkat (JEG-1) cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, penicillin (100 U/ml), and L-glutamine (2 mM). Stably transformed Jurkat and COS cell lines were obtained by transfection of cells with pSV2-neo-WZhet-expressing ZEBRA and control plasmid pSV2-neo using Cell-Fectin (Life Technologies, Gaithersburg, MD). Cells were cloned in soft agar using selection for G418 resistance (100 µg/ml) and grown in 200 µg/ml G418. EBV-immortalized B cell lines were obtained with informed consent from an EBV-positive patient as described (46). Two of three B cell lines used in this analysis expressed small amounts of BZLF-1 transcripts (17) as detected by PCR analysis (data not shown).

Luciferase assay

Reporter plasmids (0.5 µg), BZLF-1 expression plasmid and control (0.5 µg), {Delta}N1 (0.5 µg), and MEKK{Delta} (0.1 µg), were transiently transfected into 2 x 106 logarithmically growing cells with either Cell-Fectin (Life Technologies) or Superfectin (Qiagen). In some experiments, cells were also transfected with a plasmid denoted pRL-SV40 (5 ng) expressing Renilla luciferase (Promega). Cells were cultured for 36 h after transfection in RPMI 1640 medium supplemented with 10% FCS, penicillin (100 U/ml), and L-glutamine (2 mM). Luciferase activity was determined either using the firefly luciferase or Stop-and-Glo assay systems with cotransfected pRL-SV40 expressing Renilla luciferase (Promega) and an Analytical Luminescence Laboratory (San Diego, CA) luminometer. In experiments shown, luminescence was normalized to micrograms of total protein determined by Bradford protein assay (Bio-Rad, Hercules, CA).

Immunofluorescence

Cells were bound to cover slips coated with poly-D-lysine (Sigma, St. Louis, MO) and fixed with paraformaldehyde. Fixed cells were incubated with a rabbit polyclonal antisera generated against bacterially produced whole ZEBRA protein/TRPE fusion protein obtained from Dr. G. Miller or polyclonal rabbit antisera against NF-{kappa}B p50, p65, RelB, and RelC obtained from Santa Cruz Biologicals (Santa Cruz, CA). Primary antisera were used at 1/1000 dilution. Cells were washed and incubated with biotinylated donkey anti-rabbit Ab (secondary Ab), washed, and incubated with anti-streptavidin Cy3 (tertiary Ab). Secondary and tertiary Abs were obtained from Jackson ImmunoResearch (West Grove, PA) and used at 1/180 dilution. Cells were photographed using a Nikon Diaphot 60x oil immersion lens (Nikon, Melville, NY) and data were collected using IP Lab Spectrum software (Signal Analytics, Vienna, VA).

Western blotting

Whole-cell lysates for Western blotting were generated by lysis of cells in a hypertonic RIPA buffer containing 25 mM Tris, pH 7.5, 2% Nonidet P-40, 0.2% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, and 10% v/v gycerol. Abs were used at a 1/1000 dilution for Western blotting. Murine mAb OT20A recognizing ZEBRA protein was obtained from Dr. J. Middledorp (OrganonTeknika, Boxtel, Netherlands), and reactivity with ZEBRA protein was confirmed using B lymphoblastoid EBV-positive Akata cells (data not shown). EBV-reactive human antiserum was obtained with consent as described (46, 47, 48). Polyclonal rabbit antisera against NF-{kappa}B p50, p65, RelB, and RelC were obtained from Santa Cruz Biologicals (Santa Cruz, CA). A rabbit polyclonal antisera against the amino terminus of I{kappa}B{alpha} was obtained from Dr. L. Ghoda. Proteins were boiled in SDS loading buffer and separated using 12% PAGE. Western blots were developed using the Renaissance system (NEN, Boston, MA). Western blots of ZEBRA, NF-{kappa}B p50, p65, RelB, and RelC expression in Z1 and Z cells shown are representative of at least two independent experiments.

EMSA

Oligonucleotides corresponding to consensus and specific NF-{kappa}B, AP-1, and ZRE sequences are shown in Table IGo. Oligonucleotides were designed with unpaired nucleotides for subsequent fill in with [{alpha}-32P]dCTP (6000 mCi/mM; Amersham, Arlington Heights, IL) using Superscript exonuclease-minus reverse transcriptase (Life Technologies). Unincorporated nucleotides were removed with a Qiagen nucleotide removal column. One nanogram of labeled double-stranded oligonucleotide (sp. act. ~2 x 105/ng) was used in each gel shift experiment. Nuclear extracts were prepared from 2 x 106 cells as described (48). Approximately 1 µg of nuclear protein was used for each gel shift. Nuclear protein was incubated on ice for 20 min with labeled oligonucleotide in 20 µL buffer containing 20 ng/µL polyDIC (Boehringer-Mannheim), 1 mM PMSF, 1 mM DTT, 10 mM HEPES, pH 7.9, 70 mM KCl, and 2 mM MgCl2. Abs for supershift (1/100 dilution) were incubated an additional 10 min on ice after addition. Protein/oligonucleotide complexes were separated on a high-ionic strength glycine gel (49).


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  		Table I. Oligonucleotides used in this worka

 
Statistical analysis

In luciferase experiments shown, each data point was measured in triplicate. Means and SE were determined as shown graphically and analyzed using the JMP Statistical Discovery Software Version 3.1 (SAS Institute, Cary, NC). Values of p (Students t test) were determined by the JMP program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transient transfection of BZLF-1 expression plasmid decreases NF-{kappa}B transcription in HPB-ALL T lymphoblastoid cells

Experiments were designed to test the hypothesis that expression of EBV-encoded ZEBRA protein could modulate NF-{kappa}B expression in human T lymphoblastoid cells. pSV2-neo-WZhet plasmid-encoding ZEBRA or control plasmid pSV2-neo (42) were transfected transiently into HPB-ALL T lymphoblastoid cells with an NF-{kappa}B reporter gene. NF-{kappa}B transcription was simultaneously activated by cotransfection of the MEKK1 kinase (44), which directly activates the NF-{kappa}B transcription pathway (50). Under these conditions, NF-{kappa}B transcription in HPB-ALL cells was significantly decreased by cotransfection of pSV2-neo-WZhet plasmid relative to cells cotransfected with control plasmid (Fig. 1GoA). MEKK1 activation of cells in the presence or absence of pSV2-neo-WZhet plasmid did not alter transfection efficiency of cells or cell viability as assessed by cotransfection of cells with a second reporter gene expressing Renilla luciferase, and transcription of an AP-1-specific reporter gene was not activated (data not shown). Transfection of a plasmid encoding a constitutive inhibitor of I{kappa}B{alpha} ({Delta}N1) abolished MEKK1-activated NF-{kappa}B transcription either in the presence or absence of cotransfected pSV2-neo-WZhet plasmid, demonstrating the responsiveness of the reporter gene to endogenous NF-{kappa}B regulatory pathways (Fig. 1GoA). These studies established that transfection of pSV2-neo-WZhet plasmid could decrease activation of NF-{kappa}B transcription in T lymphoblastoid cells. Because MEKK1 directly activates the NF-{kappa}B-activating I{kappa}B{alpha} kinase (50), these results established that the effects of pSV2-neo-WZhet plasmid cotransfection were at or below the level of I{kappa}B{alpha} kinase activation. Similar results were evident with transient transfection of ZEBRA expression plasmid into MEKK1-activated Jurkat T lymphoblastoid cells and EBV genome-positive B lymphoblastoid cell lines, suggesting that coexpression of ZEBRA was able to block MEKK1-activated NF-{kappa}B transcription in a variety of EBV genome-negative and EBV genome-positive lymphoblastoid cells (data not shown).



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  		FIGURE 1. Expression of an NF-{kappa}B reporter gene in T lymphoblastoid cell lines HPB-ALL and Jurkat. The activity of a transfected NF-{kappa}B reporter gene is expressed as the ratio of reporter gene activity to background luciferase activity (stimulation index). A, MEKK1-activated NF-{kappa}B transcription in HPB-ALL cells was decreased (*, p < 0.05) by cotransfection of pSV2-neo-WZhet plasmid (Z+) relative to cells cotransfected with control plasmid pSV2-neo (Z-). With cotransfection of constitutive I{kappa}B{alpha} expression plasmid {Delta}N1, expression of the NF-{kappa}B reporter was eliminated both with and without cotransfection of pSV2-neo-WZhet. Pooled data from two independent experiments are shown. B, Stimulation index was not different from background or between Z- and Z+ Jurkat cells in the absence of cellular activation (MEKK-). MEKK1-activated transcription (MEKK+) of an NF-{kappa}B reporter gene was decreased (*, p < 0.05) in Jurkat cells stably expressing pSV2-neo-WZhet plasmid (Z+) relative to cells expressing control vector (Z-). Pooled data from six independent experiments in the Z+ Jurkat cell line denoted Z1 are shown.

 
Stable expression of ZEBRA protein is associated with decreased NF-{kappa}B transcription in Jurkat T lymphoblastoid cells

In transient tranfection experiments with the pSV2-neo-WZhet plasmid, ZEBRA protein expression was below the level of detection by Western blotting. Further studies were conducted with two Jurkat cell lines established in this work stably transfected with the pSV2-neo-WZhet plasmid (denoted Z1 and ZA) and a cell line stably transfected with control plasmid pSV2-neo (denoted Z-). Stable transfection of the pSV2-neo-WZhet plasmid did not alter basal NF-{kappa}B expression in Jurkat cells, but decreased activation of NF-{kappa}B by MEKK1 (Fig. 1GoB). Results are shown for the Z1 cell line, although similar results were also evident in the ZA cell line (data not shown). Low levels of ZEBRA protein were detected in Z1 and ZA Jurkat cells but not in control Z- cells by immunoblotting with ZEBRA-specific mAb OT20A (Fig. 2Go). The molecular mass of ZEBRA protein expressed in Jurkat cells, ~43 kDa, was similar to that described for ZEBRA protein encoded by pSV2-neo-WZhet in B lymphoblastoid cells (42). A human polyclonal antiserum with high titers against EBV lytic gene products (42) also detected ZEBRA protein expressed in Jurkat cells (Fig. 2Go).



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  		FIGURE 2. Jurkat cells express stable 43-kDa putative ZEBRA protein. ZEBRA was detected by Western blotting using anti-ZEBRA mAb OT20A (left) or a human polyclonal antiserum from a patient with chronic active EBV infection (right) only in cells transfected with pSV2-neo-WZhet plasmid (denoted Z+). Data from both Z+ Jurkat cell lines used in this work, ZA and Z1, respectively, are shown. A COS cell line stably transfected with pSV2-neo-WZhet plasmid (denoted COS) was used as a positive control for ZEBRA expression. To detect ZEBRA-specific bands in Z1 and ZA cell lines, overexposure of Western blots was required relative to Western blots of protein from EBV genome-positive B lymphoblastoid cell lines (data not shown), suggesting that relatively low amounts of ZEBRA protein were expressed in transfected cells.

 
ZEBRA protein could also be detected in Z1 cells by immunofluorescence and had a primarily cytoplasmic distribution (Fig. 3Go). Immunofluoresence was also used to determine expression of NF-{kappa}B proteins in cells expressing ZEBRA using the Z1 cell line (Fig. 3Go). As shown, immunostaining for the NF-{kappa}B p50 subunit was increased in Z1 cells relative to Z- cells, but other components of NF-{kappa}B including p65 and I{kappa}B{alpha} appeared to be expressed at similar levels in the two cell lines. Cells expressing ZEBRA had an abnormal morphology with adherence to culture dishes not evident in control cells under identical culture conditions and also decreased expression of the TCR (CD3) as determined by FACS (data not shown).



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  		FIGURE 3. Z1 Jurkat cells stably expressing ZEBRA protein as detected by Western blotting (denoted Z+) and control cells (denoted Z-) were analyzed by immunofluorescence using a rabbit polyclonal anti-serum for ZEBRA protein and rabbit polyclonal antisera specific for components of NF-{kappa}B (p50, p65, and I{kappa}B{alpha}). Representative staining of individual cells is shown. Cytoplasmic staining with ZEBRA Ab was evident in Z1 but not Z- cells. Increased expression of NF-{kappa}B p50 but not other components of NF-{kappa}B was also detected in Z1 cells. Cells expressing ZEBRA were also slightly larger and had a more adherent phenotype to plasticware than control cells.

 
ZEBRA expression is associated with increased levels of NF-{kappa}B p50 protein

Further studies were designed to clarify the mechanism through which stable expression of ZEBRA protein in Jurkat cells resulted in decreased expression of NF-{kappa}B (Fig. 1GoB). Inactivation of NF-{kappa}B could result from decreased levels of expressed p65 protein or p50 protein, which form the high-affinity NF-{kappa}B transcription factor. However, immunofluorescence studies (Fig. 3Go) did not support the hypothesis that expression of NF-{kappa}B proteins were decreased by ZEBRA expression. Expression of proteins of the NF-{kappa}B pathway were also characterized by Western blotting in cells expressing ZEBRA (Fig. 4Go). Consistent with immunofluorescence studies (Fig. 3Go), levels of p50 and other unidentified proteins cross-reactive with the p50 polyclonal antisera were markedly increased in cells expressing ZEBRA. Levels of RelA p65 and RelB were not different in cells expressing ZEBRA from control cells as determined by Western blotting. Levels of I{kappa}B{alpha} and RelC were also similar in Z1 and Z- cells as determined by Western blotting (data not shown). These observations suggested that decreased NF-{kappa}B activation in Z1 cells was not secondary to a deficiency or altered distribution of specific components of the NF-{kappa}B complex. Unexpectedly, ZEBRA expression was associated with increased expression or stability of the NF-{kappa}B p50 subunit.



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  		FIGURE 4. Increased expression of NF-{kappa}B p50 and other unidentified p50 cross-reactive species was detected by Western blotting with a rabbit polyclonal antiserum generated against p50 in Z1 cell lysates relative to Z- lysates. Expression of NF-{kappa}B p65 and RelB proteins detected with rabbit polyclonal antisera was similar in the Z1 Jurkat cell and Z- Jurkat cells. To control for protein loading, Western blots were performed in duplicate using the identical cell lysates.

 
ZEBRA and NF-{kappa}B proteins expressed in Z+ cells are not functional in DNA binding

Because decreased levels of NF-{kappa}B proteins did not account for inactivation of NF-{kappa}B in Z1 cells, we determined whether the NF-{kappa}B p50 protein present in increased quantities in the cytoplasm of Z+ cells (Fig. 4Go) was functional as a DNA binding protein. These studies were conducted both with a consensus high-affinity NF-{kappa}B binding site (37) and a variant NF-{kappa}B site in the IL-2 promoter (41). The variant site in the IL-2 promoter binds primarily to p50 homodimer (37) and variant NF-{kappa}B proteins such as the p50 homodimer have been shown to play a significant role in activation of other important immunoregulatory genes in T cells. Comparative studies were also conducted in EBV-immortalized B lymphocyte cell lines, which up-regulate NF-{kappa}B (Refs. 51 and 52 , reviewed in Ref. 1).

EMSA analysis using oligonucleotides generated from the NF-{kappa}B consensus site (37) or the IL-2 NF-{kappa}B site (41) demonstrated that specific NF-{kappa}B binding proteins were present in nuclear proteins from unstimulated Z- Jurkat cells but not from Z1 or ZA cells expressing ZEBRA (Fig. 5Go, A and B). p50 bound to a consensus high-affinity NF-{kappa}B oligonucleotide appeared to be associated with other NF-{kappa}B proteins including possibly RelB because an antisera against RelB disrupted the EMSA complex (Fig. 5GoA). In contrast to results using the high-affinity NF-{kappa}B oligonucleotide, the p50 homodimer was the only detectable species of NF-{kappa}B protein detected by EMSA in unstimulated Jurkat cells bound to an oligonucleotide derived from the IL-2 promoter NF-{kappa}B site (Fig. 5GoB). The EMSA complex bound to the IL-2 promoter NF-{kappa}B site migrated as a single species, was completely supershifted by an anti-p50 antisera, and was not affected by anti-RelB antisera (Fig. 5GoB). A limitation of these studies was our inability to completely identify all complexes bound to NF-{kappa}B in Jurkat cells (Fig. 5GoA); however, all complexes detected with either NF-{kappa}B oligonucleotide were significantly reduced by coexpression of ZEBRA.



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  		FIGURE 5. EMSA demonstrated that specific NF-{kappa}B protein/DNA complexes were present in nuclear proteins extracted from Z- cells but not in nuclear proteins extracted from two independent Z+ cell lines, Z1 and ZA. Results representative of at least two independent experiments are shown. A, EMSA of nuclear extracts from two independent Z+ cell lines (Z1 and ZA) and a Z- control cell line using a consensus NF-{kappa}B site (N51/N31, denoted N+; Table IGo) without addition of Ab (-) or with Ab against NF-{kappa}B p50, p65, and RelB proteins is shown. An NF-{kappa}B-specific complex partially supershifted with p50-reactive antiserum (N+/p50) but not with p65-reactive antiserum (N+/p65) was detected in Z- but not Z1 or ZA nuclear extracts. This complex was eliminated with an antiserum against RelB, suggesting that p50 and additional components of NF-{kappa}B were involved in formation of the basal complex on the NF-{kappa}B high-affinity site. B, Specific complexes did not bind a mutated NF-{kappa}B site (N56/36, denoted N-; Table IGo). A single nucleoprotein complex was evident in Z- but not in Z1 or ZA nuclear extracts bound to an NF-{kappa}B site corresponding to the human IL-2 promoter (N56/N36, denoted NIL2; Table IGo) as shown. NIL2 binding in Z- cells was completely supershifted by antiserum to NF-{kappa}B p50, but was not altered by antiserum against either NF-{kappa}B p65 or RelB (see Discussion). C, EMSA analysis of nuclear extracts used in A and B using an AP-1 consensus site oligonucleotide (AP1 A52/32; Table IGo) is shown. EMSA demonstrated similar AP-1 binding in nuclear proteins from Z1, ZA, and Z- cells. An oligonucleotide corresponding to the ZEBRA-specific ZRE site ZIIIA (Z57/37; Table IGo) demonstrated no evidence of a ZEBRA/ZIIIA complex in nuclear proteins obtained from Z1 or ZA cells. D, Nuclear proteins from three independent B cell lines (B1, B2, B3) derived from a single EBV-positive patient are shown. EMSA demonstrated similar nuclear binding in each cell line using the N+ consensus oligonucleotide (N51/N31, denoted N+; Table IGo). Binding to the N+ oligonucleotide was eliminated by a 100-fold excess of unlabeled N+ (denoted -), and was also not detected (data not shown) with a mutated NF-{kappa}B site (N56/36, denoted N-; Table IGo). B cell NF-{kappa}B complexes were almost entirely supershifted by Ab to NF-{kappa}B p50 and were partially supershifted by Ab to NF-{kappa}B p65 and other components of NF-{kappa}B including RelB.

 
The absence of detectable NF-{kappa}B EMSA complexes detected with either NF-{kappa}B oligonucleotide in Z+ Jurkat cells (Fig. 5Go, A and B) could not be explained as the result of selective nuclear protein degradation or poor nuclear protein isolation from Z1 or ZA cells. As shown, the same preparation of nuclear extracts from both Z+ and Z- cell lines demonstrated nearly identical specific binding to an AP-1 consensus oligonucleotide (Fig. 5GoC), confirming that equivalent amounts of nuclear proteins were extracted from Z- and Z+ cells. No evidence of a hybrid ZEBRA/p50 complex was evident bound to ZEBRA-specific AP-1-like ZRE element (47). denoted ZREIII (Table IGo), consistent with a cytoplasmic localization of ZEBRA.

Comparison of nuclear proteins isolated under similar conditions from three unstimulated EBV-immortalized B cell lines (Fig. 5GoD) demonstrated that the NF-{kappa}B consensus site oligonucleotide bound to much greater quantities of NF-{kappa}B binding proteins in EBV-immortalized B cells than in unstimulated Jurkat cells (Fig. 5GoA). This was evident because the ratio of EMSA complexes to free oligonucleotide probe was several orders of magnitude less in extracts from Z- Jurkat cells (Fig. 5Go, A and B) relative to this ratio in extracts from similar numbers of EBV-immortalized B cells (Fig. 5GoD).

As in Z- Jurkat cells, NF-{kappa}B complexes in unstimulated EBV-immortalized B cells detected by the consensus NF-{kappa}B oligonucleotide consisted primarily of complexes containing NF-{kappa}B p50, although in contrast to Jurkat cells, some NF-{kappa}B p65 was also evident (Fig. 5GoD). Some subtle differences were also apparent between NF-{kappa}B complexes bound to the NF-{kappa}B consensus high-affinity oligonucleotide in Z- Jurkat cells (Fig. 5GoA) and B cells (Fig. 5GoD). In particular, the electrophoretic mobility of NF-{kappa}B complexes bound to consensus oligonucleotide (Fig. 5GoA) in the absence of Ab supershift relative to mobility of the free probe was slightly less in Z- Jurkat cells in comparison to the electrophoretic mobility of similar complexes in B cells (Fig. 5GoD). These subtle differences could arise either from mass effects due to the greater quantities of NF-{kappa}B binding proteins in EBV-immortalized B cells or may reflect a more complex difference in NF-{kappa}B proteins present in Jurkat and EBV-immortalized B cells. These experiments demonstrated that the effects of ZEBRA upon NF-{kappa}B DNA binding were opposite to the effects of EBV-latent gene expression, which up-regulate NF-{kappa}B in stably transformed B cell lines (51, 52, 53, 54). Additionally, these observations demonstrated that p50 protein detected at increased levels in Jurkat cells expressing ZEBRA (Figs. 3Go and 4Go) was not functional as a DNA-binding protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we found that expression of ZEBRA protein in human T cells was associated with decreased stress-activated transcription of an NF-{kappa}B reporter gene (Fig. 1Go). ZEBRA protein expressed in Jurkat T lymphoblastoid cells (Fig. 2Go) was similar in size to ZEBRA protein expressed in B cells (42). Jurkat T lymphoblastoid cells expressing ZEBRA had an altered expression (Figs. 3Go and 4Go) and localization of NF-{kappa}B p50 protein (Fig. 5Go). These observations suggest that inactivation of NF-{kappa}B by ZEBRA protein could be a mechanism of EBV pathogenesis in human T cells, because inactivation of NF-{kappa}B in Jurkat cells previously has been shown to increase cellular apoptosis (39) and altered T cell repertoire development (40). ZEBRA appears to act in opposition to the effects of EBV latency gene products that up-regulate NF-{kappa}B transcription through constitutive activation of the TNF-{alpha} receptor complex (51, 52, 53, 54). Although other viruses have been demonstrated to encode proteins that seem to be viral copies of I{kappa}B{alpha}, in contrast to ZEBRA, these proteins do not play an essential role in the lifecycle of viruses that encode them and are not encoded by human pathogens (55).

Binding between p65 and ZEBRA could block NF-{kappa}B activation by binding to NF-{kappa}B in the cell cytoplasm and inhibiting translocation of NF-{kappa}B to the cell nucleus, or alternatively by forming nuclear complexes with DNA on NF-{kappa}B sites. Because no evidence of an intranuclear blocking complex on NF-{kappa}B sites was evident in T cells expressing ZEBRA (Fig. 5Go), it is likely that the effects of ZEBRA on NF-{kappa}B involve cytoplasmic interactions through an inhibition of NF-{kappa}B translocation to the cell nucleus. A defect in NF-{kappa}B p50 nuclear translocation could account for the observation that increased quantities of the NF-{kappa}B p50 protein are not functional in DNA binding to NF-{kappa}B sites. ZEBRA protein was also detected by immunofluorescence in the cytoplasmic fraction of Z1 cell proteins (Fig. 3Go) and did not activate transcription of an AP-1 reporter gene in unstimulated Z+ cells (AP-1 reporter data not shown) also supporting a cytoplasmic location of the ZEBRA protein expressed in Jurkat cells. No evidence of ZEBRA protein bound to a high-affinity ZRE site (ZIIIA) was evident by EMSA (Fig. 5GoC).

These observations are consistent with the role of ZEBRA as an inhibitor of NF-{kappa}B transcription through interference with the nuclear translocation of components of NF-{kappa}B, although the mechanism of this interference is not established in this work. Binding between ZEBRA and components of NF-{kappa}B could also affect posttranslational processing and lead to altered cytoplasmic concentrations of NF-{kappa}B. Notably, the endogenous inhibitor of NF-{kappa}B, I{kappa}B{alpha}, binds to both NF-{kappa}B p65 and blocks the nuclear localization signals of both proteins. Binding between I{kappa}B{alpha} and components of NF-{kappa}B can occur either in the cell cytoplasm or the cell nucleus (36).

A number of molecular mechanisms could account for the ability of ZEBRA to interfere with the activation and translocation of components of NF-{kappa}B. For example, ZEBRA could directly or indirectly block phosphorylative degradation of I{kappa}B{alpha} bound to NF-{kappa}B complexes. Alternatively, ZEBRA could compete with the binding of I{kappa}B{alpha} to NF-{kappa}B and, like I{kappa}B{alpha}, mask the nuclear localization signal on NF-{kappa}B (36). ZEBRA could also bind to an additional cellular protein such as a pore required for nuclear translocation of NF-{kappa}B. Thus ZEBRA could interfere with both activation of the stress-activated high-affinity p50/p65 heterodimer through MEKK1 activation of NF-{kappa}B (Fig. 1Go) and with basal DNA binding of NF-{kappa}B p50 (Fig. 5Go). Posttranslational modifications of ZEBRA (56) could also play a role in binding interactions with NF-{kappa}B through their effects upon the cellular localization of ZEBRA or ability to bind to components of NF-{kappa}B. It would be useful to identify the specific interactions between ZEBRA and p65 because interference with this binding could potentially block viral replication in EBV-infected cells.

Regarding the previous observation that inactivation of NF-{kappa}B can increase apoptosis of Jurkat cells (39), one of two cell lines characterized in this work (ZA cells) also demonstrated a stable phenotype of increased apoptosis and was particularly sensitive to the proapoptotic stimulus TNF-{alpha} (data not shown). Interestingly, a second Jurkat cell line (the Z1 cell line) exhibited decreased expression of an NF-{kappa}B reporter gene (Fig. 1Go) and expressed less NF-{kappa}B binding proteins detected by EMSA than Z- cells (Fig. 5Go) but did not exhibit increased apoptotic cell death relative to Z- cells. Because only one of two cell lines stably expressing ZEBRA exhibited increased apoptosis relative to control cells, the phenotype of increased apoptosis in Jurkat cells may reflect additional factors in addition to or in conjunction with inactivation of NF-{kappa}B transcription. Therefore, additional studies of Jurkat and primary T cells infected with EBV would be required to determine whether the subtle effects of ZEBRA upon NF-{kappa}B described in this work are sufficient to alter cellular apoptosis in EBV-infected T cells.

Further studies of EBV infection of T lymphocytes, particularly primary T cells infected with EBV and T lymphocytes coinfected with EBV and other T cell lymphotrophic viruses such as HIV (16), will be required to define the role of ZEBRA expression in all aspects of T cell dysfunction typical of primary EBV infection. Because T lymphocytes are required for control of EBV lytic infection, we hypothesize that NF-{kappa}B is inactivated in primary T cells infected with EBV as a mechanism of viral pathogenesis. Inactivation of NF-{kappa}B could contribute to altered cytokine synthesis by infected cells or more directly lead to apoptosis of T cells responding to EBV infection. The effects of EBV infection on T cells would thus be similar to the effects of other human herpes viruses including herpes simplex and human herpes virus 6 that trigger T cell apoptosis in cells that are not productively infected with virus (57). Relatively low levels of ZEBRA protein detected in primary thymocytes infected with EBV B95-8 virus would perhaps be misleading (17), because activated cells expressing ZEBRA would simultaneously undergo programmed cell death. Possibly, loss of T cells with high affinity for EBV Ags during primary EBV infection could lead to increased inflammation and contribute to altered cellular immunity typical of active EBV infection (2).


   Acknowledgments
 
We thank Dr. James F. Jones, Dr. Shannon Kenney, Dr. George Miller, Dr. Gary Johnson, and Dr. Lucy Ghoda for providing reagents and for helpful discussions. We also thank Dr. Avi Kupfer and Hannah Kupfer for their assistance with immunofluorescence studies. We thank Diana Nabighian for her assistance in the preparation of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Training Grant T32-AI07365 (to D.H.D.). Back

2 Address correspondence and reprint requests to Dr. Erwin W. Gelfand, National Jewish Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: Back

3 Abbreviations used in this paper: ZRE, ZEBRA response element; MEKK, mitogen-activated protein/extracellular signal-related protein kinase kinase. Back

Received for publication February 1, 1999. Accepted for publication September 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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