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Epstein–Barr virus nuclear antigen 1 activates transcription from episomal but not integrated DNA and does not alter lymphocyte growth

Abstract

By binding to a cis-acting element (oriP) in the Epstein–Barr virus (EBV) genome, EBV nuclear antigen 1 (EBNA1) enables persistence and enhances transcription from EBV episomes. To investigate whether EBNA1 also directly affects cell gene transcription, we conditionally expressed a Flag-tagged dominant negative EBNA1 (FDNE) in an EBV immortalized lymphoblastoid cell line, in which the EBV genome is integrated into cell DNA. FDNE induction inhibited expression from an EBNA1-dependent oriP reporter plasmid by more than 90% in these cells but did not affect expression from integrated EBV or oriP reporter DNA. FDNE induction also did not alter expression of more than 1,800 cellular mRNAs. Lymphoblastoid cell line growth under a variety of conditions was unaffected by FDNE induction. Although Gal4-VP16 and EBNA1 strongly activated and coactivated a Gal4-VP16- and oriP-dependent promoter that was on an episome, only Gal4-VP16 activated the promoter when it was integrated into chromosomal DNA. These data indicate that EBNA1 is specifically deficient in activation of an integrated oriP enhancer and does not affect cell growth or gene expression through an interaction with cognate chromosomal DNA.

Epstein–Barr virus (EBV) establishes latent infection in human B lymphocytes, efficiently transforms these cells into lymphoblastoid cell lines (LCLs), and is implicated in the etiology of infectious mononucleosis, polyclonal B lymphoproliferative diseases (PLD), Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), and Hodgkin's disease (HD) (for reviews, see refs. 1 and 2). In EBV-driven lymphoproliferations, such as LCLs and PLD, EBV encodes six nuclear antigens (EBNAs), two latent infection integral membrane proteins (LMPs), two small RNAs [EBV-encoded RNAs (EBERs)], and alternatively spliced BamA rightward transcripts that may encode two other proteins (3–5). However, in BL, NPC, and HD, and in most latently infected B lymphocytes in persistently infected humans, EBNA1 is expressed in the absence of other EBNAs (6).

Because EBNA1 is nearly ubiquitously expressed in latent EBV infection, including all EBV-associated malignancies (for review, see ref. 2), EBNA1 could have a role in oncogenesis. Reverse genetic evaluation of the putative role of EBNA1 in oncogenesis is difficult, because EBNA1 is essential for efficient maintenance and expression from EBV episomes in dividing cells (7, 8). EBNA1 dimers bind to cognate sites in oriP, a cis-acting element required for EBNA1-dependent episome maintenance (7, 9–11). OriP is composed of two elements, Family of Repeats (FR) and Dyad Symmetry (DS). FR consists of 20 direct repeats of a dyad symmetry of the EBNA1 cognate sequence, and DS is composed of four additional repeats of the dyad symmetry (12). DS can be an origin for episome DNA replication during S phase in human cells, and FR is an EBNA1-dependent enhancer of transcription and episome persistence (13–15). EBNA1 amino acids 1–89 and 322–379 cooperatively mediate association with cell chromosomes and are essential for episome persistence and transcription (16–19), whereas amino acids 90–327 are a glycine- and alanine-rich domain that inhibits proteasome-mediated processing (20), amino acids 379–386 are a nuclear localization sequence, amino acids 459–604 are a dimerization and DNA-binding domain that is sufficient for oriP-dependent DNA replication (9), and amino acids 605–641 include an acidic activating domain (21) (Fig. 1A).

Figure 1.

Figure 1

In A, schematic diagrams of the EBNA1 ORF, the FDNE, and the EBNA1-dependent oriP and Gal4-VP16-dependent reporter plasmids, pFL and pGFL. Basic, GA, NLS, DBD/DD, and AC refer to the basic amino acid-rich chromosome association domain, the glycine alanine-rich domain, the nuclear localization sequence, the DNA-binding and dimerization domain, and the acidic domain, respectively. In pFL and pGFL, DS, and FR refer to the dyad symmetry and family of repeats components of oriP. pFL and pGFL have a simian virus (SV)40 minimal promoter for transcription of the luciferase. The SV40 promoter initiates transcription of the puromycin acetyl transferase. In B, FDNE inhibits wild-type EBNA1 activation of luciferase expression in non-EBV-infected BJAB cells. BJAB cells were transiently cotransfected with 20 μg of pCMVEBNA1, pCMVFDNE, or pCMV, 5 μg of pFL oriP reporter, and 5 μg of pGKβ-galactosidase control DNA. In C, conditional expression of FDNE in EBV infected IB4 LCL inhibits EBNA1 activation of luciferase expression from transfected pFL plasmid. FDNE was induced or repressed for 4 days in IBDN cells, and 10 million cells were transiently cotransfected at 230 V and 65 msec with 5 μg of pFL and 2.5 μg of pGK–β-galactosidase DNA. Luciferase and β-galactosidase were assayed after 48 h. Luciferase values were corrected for transfection efficiencies as measured by β-galactosidase activity.

EBNA1 has been implicated in EBV effects on cell gene expression and growth. Transgenic EBNA1 expression in mouse B lymphocytes is associated with an increased incidence of lymphoma (22). Further, EBNA1 expression by gene transfer is associated with increased CD25 and RAG1 and -2 expression (23, 24) and with increased permissiveness for hepatitis C virus replication (25). A cell protein can also specifically recognize EBNA1 cognate DNA consistent with the possibility that EBNA1-binding sites may be present in the cell genome (26).

The experiments reported here investigate whether EBNA1 has a direct role in LCL growth and gene expression by using conditional expression of a dominant negative EBNA1 to inhibit EBNA1 effects in an LCL, which was established by infection of primary human B lymphocyte in vitro, after which the EBV genome integrated on chromosome 4 (27–30). Despite the integration of the EBV genome in IB4 cells, IB4 cells are similar to other LCLs in activation and adhesion molecule expression and in their dependence on high cell density and rich medium for continued growth (31). IB4 cell survival is stringently dependent on EBV gene expression, including LMP1 activation of NF-κB and EBNA-2 activation of c-myc transcription through RBP-Jk (ref. 32; A. Cooper and E.K., unpublished observations). Thus, the IB4 LCL is expected to depend on a putative transcriptional or growth-enhancing effect of EBNA1.

Materials and Methods

Cell Lines and Plasmids.

Cells were maintained at 37°C in complete medium at 37°C with 5% CO2 (32). IB4 with tetracycline repressible transactivator (IBTR) and BJABTR are clones of IB4 and BJAB cells that express a tetracycline repressible, otherwise transcriptionally active, mutant form of the Tet repressor fused to the VP16 acidic activation domain (32, 33). Plasmids pCMVEBNA1 and pSVEBNA1 are the filled-in TfiI site to HindIII and BamHI to HindIII fragments of EBNA1 DNA cloned into the HindllI site of pcDNA3 (Invitrogen) or the BamHI site of pSG5 (Stratagene). The plasmid pCMVFDNE expresses a Flag epitope-tagged dominant negative EBNA1 amino acids 379–641 (18). A conditional expression plasmid for Flag-tagged dominant negative EBNA1 (FDNE) (pJEFFDNE) was constructed by cloning the filled-in NdeI-HindIII FDNE fragment of pCMVFDNE into the filled-in BamHI site of pJEF4, which is 3′ to a minimal promoter with seven upstream copies of a tetracycline operator (34). The conditional EBNA1 expression plasmid, pJEFEBNA1, is the filled-in TfiI-HindIII of EBV BamHI K cloned into the filled-in BamHI site of pJEF4. The oriP-dependent luciferase reporter plasmid, pFL has the filled-in EcoRI-HpaI oriP fragment of EBV DNA cloned into the SmaI site of pGL2p (Promega). FR is proximal and DS distal to the enhancerless simian virus 40 promoter and luciferase reporter. Plasmid pFL also has the SV40 promoter and puromycin acetyl transferase ORF from the filled-in EcoRI-Clal DNA fragment of pBabepuro (35) cloned into the filled-in BamHI site of pGL2p (Fig. 1A). The plasmid pGFL is pFL with the 0.5-Kbp SpeI-Bpu101 DNA fragment between FR and DS replaced with a 0.2-Kbp XbaI-StuI that contains 9 Gal4 DNA-binding sites (Fig. 1A). The plasmid pGL has 9sp Gal4 DNA-binding sites, a synthetic TATAA, and the firefly luciferase ORF.

IBDN are IBTR cells that conditionally express the dominant negative EBNA1, FDNE, in response to tetracycline withdrawal. IBDN cells were made by electroporating 20 million IBTR cells with 20 μg of pJEFFDNE by using a BTX electroporator (BTX, San Diego). After 1 day, 1,500 cells were plated in 150 μl of complete medium with 500 μg/ml of G418 and 1 μg/ml of tetracycline in each well of 96-well plates. Clones (IBDN) and subclones (IBDN1 and -2) that express high levels of FDNE in response to tetracycline withdrawal, and little or no detectable FDNE in the presence of tetracycline were identified by immune blotting by using the EBNA1 carboxyl-terminal-specific monoclonal mouse antibody (Advanced Biotechnology, Columbia, MD). Clones of IBDN1 cells that harbor pFL were derived by electroporating 20 million IBDN1 cells with 5 μg of pFL, followed by selection in 0.5 μg/ml of puromycin in 96-well plates. Polyclonal cell lines were selected by bulk plating. Polyclonal tetracycline-repressible EBNA1-converted BJAB cell lines were made by electroporating BJABTR cells with pJEFEBNA1 and plating in complete medium with 1 mg/ml of G418 and 0.5 μg/ml of tetracycline. Polyclonal BJAB or BJABTR cells with integrated pGL, pFL, or pGFL were made by transfection with Xmnl linearized pGL or DrdI linearized pFL or pGFL followed by selection with 400 μg/ml of hygromycin or 0.5 μg/ml of puromycin for 2 weeks.

Transient Transfection, Immune Precipitations, and Reporter Assays.

Ten million BJAB cells in log phase were electroporated with 10 μg of pCMVEBNA1 and 10 μg of pCMVFDNE or pCMVFKID, as a noninteracting negative control. After 48 h, cells were lysed in 1 ml of 0.5% Nonidet P-40/150 mM NaCl/1.5 mM EDTA/3% glycerol/50 mM Tris⋅Cl, pH7.4, with phenylmethylsulfonyl fluoride and leupeptin. Immune precipitation was by rotation with 2.5 μl of anti-EBNA1 antibody and 25 μl of protein G agarose (Amersham Pharmacia–Pharmacia Biotech) or anti-Flag (M2) bead for 2 h at 4°C (Sigma). Luciferase and β-galactosidase were assayed by using an OPTOCOMP-1 luminometer (MGM Instruments, Hamden, CT) and illuminescent reagents (Promega).

Transactivation and Episome Maintenance Assay in Vivo.

IBDN cells that have episomal or integrated pFL were grown in 0.5 μg/ml of tetracycline for 15 days, washed three times with warm complete media, seeded into warm complete media in two flasks differing in the presence or absence of tetracycline, and assayed daily for luciferase. Episome retention was assayed daily by counting viable cells in medium with 0.5 μg/ml of puromycin.

Cell Growth and Gene Expression.

IBDN- or BJAB-inducible EBNA1 cells were seeded at 100,000–200,000 cells/ml in RPMI 1640 medium with 10, 5, 2, or 1% serum and 0 or 0.5 μg/ml of tetracycline and counted daily. Protein levels from 2 × 105 cells were assayed by immune blot of whole cell extracts with EBNA1, p53, Bcl-2, or mdm2 specific antibodies (Santa Cruz), or EBV immune human serum. After 10 days, total RNAs were isolated by using RNAZOL-B (Tel-Test, Newark, NJ), and evaluated by Northern blot with GAPDH probe. First-stranded cDNA probes were synthesized from 5 μg of total RNA by using Superscript II RT, 33P-dCTP, and oligo dT primer (GIBCO/BRL) and purified by Probe Quant G50 Micro Columns (Amersham Pharmacia–Biotech). Probes were boiled, added to hybridization buffer, and incubated overnight at 42°C in 1 ml of Microhyb solution with 5 μl of boiled Cot-1 DNA and 25 μl of polydA with Human Named Gene Filter (GF211) containing 4,100 human cDNAs of known function (Research Genetics, Huntsville, AL). Filters were washed, developed overnight in a PhosphorImager cassette, and evaluated by using phosphorimager si, imagequant (Molecular Dynamics), and pscan1.1 (Research Genetics). Each 2-fold change with FDNE induction or repression was visually inspected to minimize potential artifacts.

Results

FDNE Associates with EBNA1 and Inhibits oriP Transactivation.

A Flag epitope-tagged dominant negative EBNA1, FDNE, composed of EBNA1 379–641 aa that is similar to a 450–641-aa dominant negative EBNA1 (36) was tested for blocking EBNA1 activation of an oriP-dependent luciferase reporter in BJAB, a non-EBV-infected BL cell line. EBNA1 activated a cotransfected oriP-dependent luciferase reporter on pFL plasmid DNA ≈122-fold, whereas an FDNE expression vector activated luciferase less than 2-fold (Fig. 1 A and B and data not shown). Transfection with equal amounts of EBNA1 and FDNE expression plasmids resulted in 5- to 6-fold luciferase activation indicative of an 95% inhibition of the 122-fold EBNA1 effect (Fig. 1B). FDNE and EBNA1 were expressed at similar levels in immune blots by using a monoclonal antibody that detects an epitope common to both proteins (data not shown). Immune precipitation of Flag epitope tagged FDNE proteins with M2 Flag epitope specific antibody from lysates of cells transfected with equal amounts of vectors expressing nontagged EBNA1 and FDNE, resulted in the coimmune precipitation of similar amounts of FDNE and EBNA1 (data not shown). These data indicate that FDNE dimerizes with EBNA1 and inhibits EBNA1 activation of oriP-dependent luciferase expression.

Regulated FDNE Overexpression in IB4 Cells Does Not Alter Latent EBV Gene Expression or Cell Growth but Inhibits Expression and Persistence of oriP Episomes.

A cell line, IBDN, and subclones, IBDN1 and -2, that conditionally express FDNE were derived from the IB4 LCL under conditions of FDNE repression. IBDN1 and -2 express ≈5-fold more FDNE than endogenous EBNA1 after withdrawal of tetracycline from the medium (Fig. 2). IBDN, -1, and -2 cells grew similarly in complete medium, under conditions of FDNE induction or repression, providing an initial indication that EBNA1 might be relatively unimportant for continued IB4 cell growth.

Figure 2.

Figure 2

The effects of FDNE induction or repression on EBV-encoded or cell gene expression. IBDN1 and -2, subclones of IB4 LCLs with conditional FDNE expression, were plated for 4 days in complete medium with or without tetracycline to repress (−) or induce (+) FDNE, respectively. Whole cell Western blots from 2 × 105 cells were done for EBNA1 and FDNE by using an EBNA1 monoclonal antibody, for all EBNAs by using an EBV immune human serum, and for c-myc by using a polyclonal rabbit antiserum. EBER and GAPDH expression was evaluated by Northern blot. A whole cell Western blot of BJAB cells that express FDNE is shown in the left lane.

Immune blots evaluating the effect of FDNE induction on IBDN cell EBV gene expression indicated that EBNA-LP, -2, -3A, and -3C and LMP1, which are critical for LCL growth and survival, are expressed at similar levels under conditions of FDNE induction or repression (Fig. 2 and data not shown). This was a surprising result, because EBNA1 has been implicated in activation of the EBNA and LMP1 promoters on plasmid DNAs that contain oriP (13, 37). A small effect on expression of EBERs was consistently noted; EBERs decreased by less than 50% at days 3, 7, 13, and 24 after FDNE induction (Figs. 2 and 4B and data not shown). The EBER genes are 0.5 kbp from FR and are transcribed by RNA polymerases III and II (38).

Figure 4.

Figure 4

Dominant negative FDNE induction or repression does not affect LCL growth or gene expression. In A, the growth of IBDN cells in medium supplemented with 2% serum was unaffected by FDNE induction (open circles) or repression (closed squares) over 11 days. FDNE levels as assayed by Western blot are shown below the growth. In B are Northern analyses with 32P-labeled GAPDH and EBER DNA probes of total RNA from cells 10 days after FDNE induction or repression. In C, the effect of FDNE induction for 10 days on cell gene RNA expression was evaluated by comparing RNA abundance as assayed on ≈4,100 human cDNAs under conditions of FDNE induction (EI + FDNE) and FDNE repression (EI). The ordinate and abscissa are in log base 2. The box demarcates genes whose expression is less than 2 times the median background and therefore deemed to be insignificantly different from background. The results shown are from one experiment, and the few RNAs that appear to be differentially expressed were not differentially expressed in other two experiments.

To directly assess FDNE effects on EBNA1-oriP transcriptional enhancement in IBDN cells, pFL was transfected into FDNE-induced or -repressed IBDN cells and luciferase was assayed 2 days later (Fig. 1 A and C). Luciferase activity in IBDN cells under FDNE-induced conditions was about 9% that under repressed conditions (Fig. 1C). β-Galactosidase expression from a cotransfected EBNA1-independent oriP-negative control plasmid was similar, under conditions of FDNE induction and repression. Thus FDNE induction blocked 91% of the EBNA1–oriP-dependent luciferase expression from plasmid DNA in IBDN cells.

Although these results indicate that FDNE substantially inhibits EBNA1 activation of oriP-dependent transcription from a plasmid that has been introduced into cells in which FDNE and EBNA1 are already expressed, they do not exclude the possibility that FDNE may be unable to displace EBNA1 from a plasmid that was already loaded with EBNA1. To evaluate this possibility, clones of IBDN cells were derived that stably harbor episomal or integrated pFL DNA by in situ cell lysis gel analyses (ref. 39 and data not shown), and the effect of FDNE induction was assayed. FDNE was not detectable before induction and was maximally induced by day 3 (Fig. 3A). Luciferase reporter activity of cells with episomal pFL DNA was reduced 70% by day 3 and more than 90% by day 7. In sharp contrast, luciferase activity in IBDN cells with integrated pFL did not decrease under conditions of FDNE induction (Fig. 3A). Similar data were obtained with polyclonal integrated pFL in IBDN cells and with multiple independent clones of pFL integrated into IBDN cells. Over 7 days, the effect of FDNE induction on luciferase activity in IBDN cells with episomal pFL correlated with the loss of puromycin resistance, which is indicative of the loss of oriP episomes (Fig. 3B). These data indicate that FDNE induction in IBDN cells inhibits EBNA1 effects on preexisting episomes but does not inhibit expression from an oriP element that is integrated into cell DNA.

Figure 3.

Figure 3

FDNE induction inhibits EBNA1-dependent maintenance of and expression from pFL episome but does not affect expression from integrated pFL. In A, luciferase activity per 40,000 cells for each day of FDNE induction is indicated relative to the activity per 40,000 cells of the same clone grown under conditions of FDNE repression in parallel over 7 days. A subclone of IBDN1 cells with episomal pFL was compared with 11 subclones of IBDN cells with integrated pFL DNA. Representative data from triplicate experiments are shown. FDNE expression was assayed by Western blot, and results shown are for cells grown under induced conditions relative to the repressed condition at day 0 before induction. In B, the clone of IBDN cells with episomal pFL was grown under conditions of puromycin selection and FDNE induction (open circle) or repression (closed circle), and the number of viable cells was counted each day.

FDNE Induction in IBDN Cells Does Not Alter Cell Growth or Gene Expression.

To formally assess whether EBNA1 activity on cognate DNA in IBDN cells under conditions of FDNE induction correlates with an effect on cell growth, the growth and survival of IBDN cells, induced or repressed for FDNE expression, was assayed under a range of maximal through minimal growth conditions. FDNE-induced and -repressed IBDN cells were identical in their growth when plated at 100,000 cells per ml of RPMI 1640 medium supplemented with 10, 2, 1, 0.5, or 0.1% FCS. Medium supplemented with 10% FCS gave vigorous cell growth with cells doubling every 24–36 h, whereas 2, 1, 0.5, or 0.1% serum enabled progressively less active cell growth (Fig. 4A and data not shown). Similar results were obtained with conditional EBNA1 expression at LCL levels in BL41 or BJAB non-EBV-infected BL lymphoblasts (data not shown). EBNA1 did not alter BL41 or BJAB cell growth in medium supplemented with 1 or 10% FCS. Thus, EBNA1 has no apparent effect on the growth or survival of BL cells or IB4 LCL.

Transcriptional profiling of cell gene expression was undertaken to more broadly assess the effect of FDNE induction on EBNA1 activation of cell gene expression. IBDN cells with pFL episomes were grown for 10 days in log phase in complete medium under conditions of FDNE induction or repression, as shown by Western blot in Fig. 4A. At day 10, EBNA1–oriP-dependent luciferase activity in FDNE-induced cells was less than 10% that of FDNE-repressed control cells, indicating that FDNE was functional. RNA was harvested in two different experiments. By Northern blot, EBER RNA was reduced in FDNE-induced versus -repressed cells, whereas GAPDH mRNA was intact and unaffected (Fig. 4B). Three pairs of 33P-labeled cDNA probes were prepared from the FDNE-induced and -repressed cell RNA preparations and hybridized to filters with arrays of ≈4,100 human cDNAs of known or imputed function (Research Genetics). RAG1, RAG2, and CD25 cDNAs were not arrayed on the filter. The hybridization of each 33P-labeled cDNA probe from FDNE-induced cell RNA to the arrayed cDNAs corrected for the median hybridization was compared with the hybridization of the parallel 33P-labeled cDNA probe from RNA of the same culture grown under FDNE repressed conditions, hybridized to the same filter, and corrected for the median hybridization. The ratio of FNDE repressed to induced or induced to repressed probe hybridization to any specific cell cDNA was not greater than two in more than one of three comparisons (Fig. 4C and data not shown). Thus, FDNE expression at a high level did not alter expression of any of the more than ≈1,800 cDNAs for which there was hybridization above background.

FDNE induction in IBDN cells also had no effect on expression of c-Myc, p53, Bcl-2, and MDM2 proteins, as assessed by immune blot with specific antisera (Fig. 2 and data not shown).

EBNA1 Activates Expression from oriP-Containing Episomes but Not from Integrated DNA.

The failure of FDNE induction to inhibit gene expression from integrated EBV DNA in IBDN cells or to inhibit luciferase expression from integrated pFL DNA supports the hypothesis that EBNA1 has minimal effects on integrated oriP-dependent gene expression. To directly investigate whether EBNA1 is able to activate transcription from integrated pFL DNA, two polyclonal populations of cells with integrated linearized pFL DNA were derived in the background of BJAB BL cells that conditionally expresses EBNA1 at the same level as EBV LCLs. Luciferase expression from both polyclonal BJAB cells with integrated pFL DNA was unaffected by the induction of EBNA1 expression or by transfection with an EBNA1 expression plasmid, despite EBNA1 expression in at least 50% of the cells in the latter experiments (Fig. 5A and data not shown). Thus, EBNA1 did not activate luciferase expression through an oriP enhancer in integrated pFL DNA.

Figure 5.

Figure 5

EBNA1 activates an EBNA1-dependent oriP episome enhancer on plasmids but not integrated into chromosomal DNA, whereas Gal4-VP16 activates a Gal4 response element on plasmids or integrated into chromosomal DNA. A shows the relative luciferase activities of 40,000 cells from a polyclonal BJAB cell line with integrated pFL grown under conditions of EBNA1 induction or repression. B shows Gal4-VP16 expression vector-mediated activation of luciferase expression from cotransfected pGL plasmid or integrated linearized pGL plasmid DNA in BJAB cells relative to expression vector control DNA. For Gal4-VP16 activation of pGL plasmid DNA, BJAB cells were cotransfected with 2 μg of pGL, 10 μg of pGal4-VP16, or vector, and 2.5 μg of pGKβ-galactosidase. For Gal4-VP16 activation of integrated DNA, polyclonal BJAB cell lines with integrated pGL DNA were transfected with 10 μg of pGal4-VP16 or vector DNA and 2.5 μg of pGKβ-galactosidase DNA. Luciferase activities are normalized for β-galactosidase and for activity with expression vector control. C shows EBNA1 and Gal4-VP16 activation of luciferase expression from pGFL plasmid or integrated DNA in polyclonal BJAB cells that have integrated linearized pGFL DNA. For EBNA1 or Gal4-VP16 activation of integrated pGFL luciferase reporter DNA, cells were cotransfected with 25 μg of pSVEBNA1, 25 μg of pGal4-VP16, and/or vector DNA to constitute 50 μg of total DNA. Luciferase activities of 2 × 105 live cells were assayed at 48 h after transfection. The relative activity is normalized for vector alone. For activation from pGFL episomal plasmid DNA, the BJAB cells with integrated linearized pGFL DNA were cotransfected with 2 μg of pGFL and 2 μg of pGKβ-galactosidase DNA in addition to the 25 μg of pSVEBNA1, pGal4-VP16, or vector control DNA. The luciferase activities attributable to integrated pGFL DNA assayed in parallel were subtracted from the resulting luciferase activities, which were then normalized for the similar levels of β-galactosidase activity that were obtained and for activity with vector DNA. Data shown are the average of three experiments.

In control experiments, Gal4-VP16 activated luciferase expression from pGL DNA, which has a luciferase ORF downstream of a minimal promoter with nine upstream Gal4-binding sites, in two polyclonal BJAB cell lines with stably integrated pGL DNA, or in BJAB cells transfected with pGL plasmid DNA. Cotransfection of a Gal4-VP16 expression plasmid with pGL plasmid DNA into BJAB cells activated luciferase expression ≈150 fold, and similar levels of luciferase activation were achieved by transfection of the Gal4-VP16 expression into polyclonal BJAB cell lines that had stably integrated linearized pGL DNA (Fig. 5B). These data indicate that Gal4-VP16 differs from EBNA1 in being able to activate luciferase expression through a Gal4 DNA-binding site enhancer in plasmid or a chromosomally integrated pGL DNA in BJAB cells.

To further investigate EBNA1 versus Gal4-VP16 activation of oriP and Gal4 DNA-binding site enhancers that are on plasmids versus integrated into cell DNA, pGFL was made by inserting a DNA fragment with nine Gal4-binding sites between DS and FR in pFL (Fig. 1A). Transfection of polyclonal BJAB cells that have integrated pGFL DNA with EBNA1 or Gal4-VP16 expression vectors resulted in no induction of luciferase by EBNA1 and a 6-fold induction by Gal4-VP16 in three separate experiments (Fig. 5C). Cotransfection with the same amounts of EBNA1 and Gal4-VP16 expression vectors resulted in a slight negative effect of EBNA1 on Gal4-VP16 activation of luciferase expression (Fig. 5C). Cotransfection of the BJAB cells that have integrated linearized pGFL DNA with EBNA1 or GAL4-VP16 expression vectors and pGFL plasmid DNA resulted in 15- to 17-fold increased luciferase activity from the pGFL plasmid DNA (Fig. 5C). In comparison, cotransfection with similar amounts of EBNA1 and Gal4-VP16 expression vector resulted in 80-fold increased luciferase activity, indicating that EBNA1 and Gal4-VP16 can synergistically coactivate through neighboring cognate DNAs on episomal plasmid DNA (Fig. 5C). These data indicate that Gal4-VP16 can activate transcription from episomal plasmid or integrated linear pGFL DNA and that EBNA1 and Gal4-VP16 can coactivate transcription from pGFL episomal plasmid DNA. However, EBNA1 can neither activate nor coactivate through the FR enhancer in integrated pGFL DNA. Activation of adjacent Gal4 DNA-binding sites with GAL4-VP16 did not overcome the effect of integration in blocking EBNA1 activation of an oriP enhancer.

Discussion

These experiments were initiated to examine the effect of conditional expression of a dominant negative EBNA1 on LCL gene expression and cell growth by using the IB4 LCL, in which the entire EBV genome is integrated in tandem on chromosome 4 and has not persisted as an episome (28–30). The objective was to test whether EBNA1 has a role in continued LCL growth that is distinct from the role of EBNA1 in EBV episome maintenance and in enhanced transcription from the EBV episome. The EBNA1 dominant negative, FDNE, is EBNA1 amino acids 379–641, which includes the dimerization, DNA-binding, and nuclear localization domains (11, 21) but lacks EBNA1 amino acids 8–378, which are critical for transcriptional activation (16, 17). A smaller construct of the EBNA1 dimerization and DNA-binding domain was previously shown to have a dominant negative effect on EBNA1 activation of transcription (36). FDNE dimerizes efficiently with wild-type EBNA1, binds to cognate DNA, is deficient in transcriptional enhancement, and effectively competes with wild-type EBNA1 for enhancer occupancy. FDNE has a significant fraction of the oriP DNA replication effect of EBNA1 (18), but EBNA1 amino acids 1–378 is also required for transcriptional enhancement and episome persistence (16–18).

FDNE effectively inhibits EBNA1 activation of oriP-dependent transcription from a plasmid that is introduced into IB4 cells after induction of FDNE expression. Despite FDNE down-regulation of EBNA1-mediated transcriptional effects on cognate DNA in IB4 cells, cell growth and survival were unaffected under restrictive or optimal growth conditions. The expression of more than 1,800 cell genes was also unaffected in cDNA array analyses. Moreover, EBNA1 expression at LCL levels in BJAB or BL41 BL lymphoblasts did not increase cell growth or survival under restrictive or optimal growth conditions. These data strongly support the concept that EBNA1 does not have a significant effect on B lymphoblast cell gene expression or growth under in vitro growth conditions.

Our experiments address most specifically the possibility that EBNA1 might interact with cognate sites in cell DNA to activate transcription of specific LCL cell genes that affect cell growth or survival. EBNA1-mediated transcriptional enhancement was known to require multiple copies of dimeric EBNA1 cognate DNA for efficient activation (40), and EBNA1-binding sites have not been detectable in cell DNA by using EBNA1 affinity chromatography (41). We have now found that EBNA1 is specifically deficient in activation of even a wild-type oriP enhancer when the enhancer is integrated into cell DNA, despite robust effects on the same enhancer when the enhancer is on a plasmid in the same cells. Consistent with the lack of effect of EBNA1 on expression from integrated cognate DNA, FDNE had minimal or no effect on latent EBV gene or cell gene expression. Further, EBNA1's inability to activate transcription from cognate DNA that is integrated into lymphoblast DNA is in striking contrast to Gal4-VP16, which activates transcription from episomal plasmid or integrated DNA in the same cells. Altogether, these data make it highly unlikely that EBNA1 would activate transcription of cell genes by interaction with cognate DNA.

The silencing effect on EBNA1 activation of the oriP enhancer that is caused by integration into cell DNA could be because of differences between episome and integrated DNA in long-range repression, chromatin structure, histone deacetylation, DNA methylation, or DNA structure. The effect is unique to EBNA1, and Gal4-VP16 activation was not silenced. Gal4-VP16 activated the same promoter through adjacent sites positioned upstream of oriP FR. Unlike the VP16 acidic activation domain and functionally equivalent domains in other transactivators, EBNA1 does not activate transcription when fused wholly or in part to the Gal4 DNA-binding domain (21). The EBNA1 carboxyl-terminal acidic domain can have a weak direct effect on reporter plasmids in the context of another specific gene fusion, but deletion of this domain has only a small effect on EBNA1 enhancement of transcription (21). The VP16 and EBNA2 acidic domains have multiple interactions with basal and activated transcription factors and specifically recruit p300/CBP to directly and indirectly acetylate histone; EBNA1 may be deficient in these activities (42–47). However, EBNA1 coactivated transcription with Gal4-VP16 on plasmid DNA but did not coactivate with Gal4-Vp16 on integrated DNA. The failure of Gal4-VP16 to potentiate EBNA1 coactivation of an integrated FR enhancer is surprising and supports the hypothesis that EBNA1 activation of integrated FR requires an EBNA1-oriP enhancement function that is not relieved by Gal4-Vp16 activation at the same promoter.

Previous evidence implicating EBNA1 in EBV effects on cell growth includes an increased occurrence of B lymphocyte hyperplasia and lymphoma in two lineages of EBNA1 transgenic mice (22) and increased expression of CD25 or RAG genes in EBNA1-expressing cells (23, 24). Our observations that FDNE and EBNA1 had no discernible effects on lymphocyte gene transcription or growth, in vitro, raise the possibility that the previous effects may have been a consequence of other genetic events. However, although EBNA1 is unlikely to interact with cognate cell DNA to affect cell gene expression or growth, we cannot dismiss the possibility that EBNA1 may interact with cell proteins and affect cells through pathways that may not have been detectable in the experiments described here. EBNA1 N-terminal residues have been implicated in interaction with the cellular proteins p32 and EBP2, and interaction with EBP2 is important in episome persistence (48, 49). Also, EBNA1 amino acids 91–321 are a glycine–alanine repeat domain that can inhibit proteasome-mediated degradation of EBNA1 (20). Although evidence to date indicates that the processing of other proteins is not affected by EBNA1, protein degradation is critical to cell growth control, and an effect on catabolism of a critical regulatory protein cannot be excluded.

Acknowledgments

This research was supported by the National Cancer Institute of the U.S. Public Health Service through Grant No. CA00446 and by Korea Research Foundation Program Year 1997. Bo Zhao, George Mosialos, Ken Izumi, Ellen C. McFarland, and Kara Carter provided materials, advice, or assistance in RNA profiling and data analysis.

Abbreviations

EBV

Epstein–Barr virus

LCL

lymphoblastoid cell lines

EBNA

EBV nuclear antigen

LMP

latent infection membrane protein

EBERs

EBV-encoded RNAs

FDNE

Flag-tagged dominant negative EBNA1

BL

Burkitt's Lymphoma

FR

Family of Repeats

DS

Dyad Symmetry

References