INTRODUCTION

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Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis (12, 42) and is associated with several malignancies, including Hodgkin's disease, gastric carcinoma, and B-cell lymphomas in immunocompromised individuals (9, 18, 23, 26, 51). EBV also has been found to contribute causally to Burkitt's lymphoma and nasopharyngeal carcinoma in prospective epidemiological surveys (10, 17, 31). EBV's ability to induce and maintain proliferation of the B cells it infects likely underlies its contribution to these malignancies. Within these latently infected cells, the viral genome is present as a circular plasmid that is synthesized only once per cell cycle during S-phase (1, 61) and is efficiently partitioned to daughter cells (29, 36, 55, 56), akin to the genome of its host cell. We use the term replication to encompass these DNA synthesis and partitioning events. Only two viral components are required for the replication of the EBV genome, the latent origin oriP and its binding protein EBNA-1; all else is contributed by the cell (40, 59, 62).

oriP is composed of two cis-acting elements, the family of repeats (FR) and the region of dyad symmetry (DS), which are separated by approximately 1 kbp of DNA. The FR contains 20 imperfect copies of a 30-bp repeat to which EBNA-1 binds site specifically and with high affinity (5, 6, 44). The DS, which contains four low-affinity EBNA-1-binding sites, is the site at or near which DNA synthesis initiates (15). The FR, in conjunction with EBNA-1, is thought to ensure faithful partitioning (or maintenance) of oriP replicons. This supposition is based partially on the findings that (i) plasmids lacking 14 or more EBNA-1 binding sites in the FR of oriP fail to support long-term replication in D98/Raji and Raji cells (8, 47, 57) and (ii) addition of the FR to a plasmid containing mammalian or viral autonomously replicating sequences allows long-term replication in EBNA-1-positive cells (30, 34).

Plasmids containing oriP support efficient replication in EBNA-1-positive cells selected to retain them, being lost at a rate of 2 to 4% per cell generation after removal of selection (29, 56), a rate of loss which resembles that of ARS/CEN plasmids in Saccharomyces cerevisiae (33). We refer to these plasmids as "established" replicons in that they support efficient DNA synthesis and partitioning each cell cycle. Unexpectedly we have found that upon introduction of oriP plasmids into a population of EBNA-1-positive cells, oriP plasmids replicate but are lost precipitously from cells during 2 weeks posttransfection (>25% rate of loss per cell generation) (37). Upon investigation of these disparate observations, we have found that oriP replicons must be modified epigenetically for establishment, and this modification occurs in only 1 to 10% of transfected cells (37). Our observations of oriP plasmids are consistent with studies on EBV that demonstrate that EBV DNA is lost rapidly from proliferating cells following infection (45) and is established in approximately 1% of infected cells (27, 58).

We wished to gain insights into the cis-acting requirements for the establishment of an oriP replicon in different cell lines. We therefore monitored the replication levels of oriP plasmid derivatives in multiple EBNA-1-positive cell lines over several weeks following their introduction into cells. Here we report that cell lines differ in their ability to support the replication of a plasmid containing only the DS of oriP. In 143B and H1299 cells that express EBNA-1, DS plasmids support replication, albeit inefficiently, and are lost more rapidly from cells than are oriP plasmids. That is, EBNA-1-binding sites within the FR clearly stimulate replication in 143B and H1299 cells. Unexpectedly, however, plasmids containing only the DS of oriP were established efficiently in 293/EBNA-1 cells, providing efficient DNA synthetic and partitioning functions over 24 days posttransfection. Plasmids containing the FR in addition to the DS supported replication but were lost precipitously in 293/EBNA-1 cells and all other cell lines analyzed (27, 37, 45, 58), indicating that the FR inhibits establishment of oriP replicons in 293/EBNA-1 cells and likely inhibits establishment of oriP replicons in all other cell lines. We show that this inefficient establishment of oriP is not due to FR's enhancement of transcription from the oriP replicon or to DNA looping between the FR and DS.

In addition, the FR's ability to act as a replication fork barrier does not solely underlie the inefficient establishment of oriP replicons. Deletion of EBNA-1 binding sites within the FR or substitution of the FR with DS sequences, however, overcame the inhibitory activity of the FR, allowing efficient establishment of the oriP plasmid derivative in 293/EBNA-1 cells. Our findings indicate that the FR of oriP possesses inhibitory as well as stimulatory activities with respect to replication. By defining the mechanism by which an epigenetic event overcomes the inhibitory activity of the FR, therapeutic agents may be developed to prevent the establishment of EBV's plasmid replicon.

	MATERIALS AND METHODS

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Plasmids. 2278, the oriP test plasmid, and 2276, the prokaryotic backbone plasmid, are derived from 2275, a plasmid containing a ColE1 origin and supF marker for propagation in Escherichia coli and a neomycin phosphotransferase gene driven by the thymidine kinase promoter of herpes simplex virus. 2278 was constructed by inserting oriP between the HpaI and NsiI sites of 2275. 2276 was constructed by replacing the neomycin phosphotransferase gene with the firefly luciferase gene, generating a length polymorphism upon amplification with primers 1588 and 86, as described below. 2402.7, the test plasmid containing only the DS of oriP, was constructed by inserting the DS between the EcoRV and SalI sites of 2275. The test plasmid lacking the DS of oriP, 2331, was constructed by deleting DS from 2278 by digestion with EcoRV and religation.

Cell lines and transfections. The cell lines used for the replication assays include 143B, a human osteosarcoma cell line (ATCC CRL-8303); C33A/EBNA-1, a human cervical carcinoma cell line (ATCC HTB-31) into which plasmid 1553, which expresses EBNA-1 and hygromycin B phosphotransferase, was integrated (2); H1299/1728#3, a p53-null human lung carcinoma cell line (ATCC CRL-5803) that maintains the 1728 oriP/EBNA-1 expression plasmid (37); 293/EBNA-1, a human embryonic kidney cell line that stably expresses EBNA-1 and neomycin phosphotransferase (ATCC CRL 10852); and the 293/1728#5 cell clone, which was selected to stably replicate the 1728 oriP/EBNA-1 expression plasmid (37). Cell lines were grown in Dulbecco's modified Eagle's medium with high glucose and supplemented with 10% fetal bovine serum and 200 U of penicillin and 200 µg of streptomycin sulfate per ml. 143B cells were grown in medium containing calf serum instead of fetal bovine serum. 293/EBNA-1, C33A/EBNA-1, H1299/1728#3, and 293/1728#5 cell lines were also grown in the presence of G418 sulfate (200 µg/ml) and 100, 300, and 200 µg of hygromycin B/ml, respectively. Cells were grown at 37°C in a humidified 5% CO₂ atmosphere. The doubling time of these cell lines is 22 to 24 h.

Quantitative competitive PCR assay. At the indicated time points posttransfection, cells were harvested, and low-molecular-weight DNA was extracted by the method of Hirt (25) and prepared as described (37).

Cesium chloride density gradient analysis. Equimolar amounts of an oriP plasmid (10 µg of oriP) and a plasmid containing only the DS of oriP (6.5 µg of DS) were introduced independently into 293/EBNA-1 cells together with 5 µg of 2145 (an expression vector for EGFP). At 2 days posttransfection, cells were harvested and the percent EGFP-positive cells was enumerated as a measure of the transfection efficiency. For each transfection, approximately 50% of the cells were EGFP positive. The cells were expanded on 15-cm dishes and labeled with 100 µM bromodeoxyuridine and 200 µM deoxycytidine for 17 h at 5 days posttransfection (61). Plasmid DNA was isolated by Hirt extraction of approximately 5 × 10⁷ cells (as described above), linearized with XhoI, and mixed with CsCl to a density of 1.74 g/ml in a 5-ml volume (61). These samples were spun at 60,000 rpm for 19 h in a Beckman type NVT 65.2 rotor and fractionated into 200-µl aliquots, and the refractive index of every fourth fraction was determined. One fourth of each fraction was denatured, transferred to Gene Screen Plus hybridization membrane (NEN Life Sciences) via dot-blotting (Schleicher and Schuell Minifold I), and hybridized to labeled DNAs encompassing the neomycin phosphotransferase gene. Signals were captured with a phosphorimager and analyzed with ImageQuant software (Molecular Dynamics).

	RESULTS

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Plasmids containing only DS of oriP support replication with various efficiencies in different cell lines. To gain insight into the cis-acting requirements for the establishment of an oriP replicon in different cell lines, we monitored the replication levels of oriP plasmid derivatives containing wild-type oriP (oriP), only the DS of oriP (DS), and only the FR of oriP (FR) in multiple EBNA-1-positive cell lines at early times posttransfection. The fate of these replicated plasmids was then monitored over 2 weeks following their introduction into cells. To do this, equimolar amounts of a test plasmid (oriP, DS, or FR) and prokaryotic backbone plasmid (which serves as an internal negative control) were introduced into 143B cells with or without an expression plasmid for EBNA-1 or into C33A/EBNA-1, H1299/1728#3, and 293/EBNA-1 cells that stably express EBNA-1. Cells were grown in the absence of selection. At different times following transfection, plasmid DNA was isolated by Hirt extraction and digested exhaustively with DpnI, and the level of replicated, DpnI-resistant DNA was determined by quantitative competitive PCR (28).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Plasmid containing only the DS of oriP supports EBNA-1-dependent replication, albeit inefficienty, at 4 days posttransfection in 143B cells. A test plasmid containing oriP (oriP) or only the DS of oriP (DS) was introduced into 143B cells together with an equimolar amount of prokaryotic backbone plasmid (Backbone) either with (+EBNA-1) or without (EBNA-1) an expression plasmid for EBNA-1. At 4 days posttransfection, plasmid DNA was isolated by Hirt extraction and digested with XhoI and DpnI, and the level of replicated, DpnI-resistant DNA was determined by quantitative competitive PCR. PCRs were performed using 6 × 105 cell equivalents and a competitor DNA standard curve (9 pg, 3 pg, 600 fg, 120 fg, and 24 fg). Numbers below each gel refer to the average number of molecules present per transfected cell.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   Plasmid containing only the DS of oriP replicates within C33A/EBNA-1 cells but is lost precipitously during 2 weeks posttransfection, as is the oriP plasmid. Equimolar amounts of an oriP test plasmid (oriP), a test plasmid containing only the DS of oriP (DS), and a test plasmid containing only the FR of oriP (FR) were introduced independently into C33A/EBNA-1 cells together with a prokaryotic backbone plasmid (Backbone). At the indicated times posttransfection, plasmid DNA was isolated by Hirt extraction and digested with XhoI and DpnI, and the level of replicated, DpnI-resistant DNA was determined by quantitative competitive PCR. PCRs were performed using 105 cell equivalents and a competitor DNA standard curve (9 pg, 3 pg, 600 fg, 120 fg, and 24 fg). Numbers below each gel refer to the average number of molecules present per transfected cell.6d., day 6 posttransfection.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   (A) Plasmid containing only the DS of oriP supports efficient replication over 16 days posttransfection in 293/EBNA-1 cells, while replicated oriP plasmids are lost precipitously. Equimolar amounts of an oriP test plasmid (oriP), a test plasmid containing only the DS of oriP (DS), and a test plasmid lacking the DS of oriP (FR) were introduced independently into 293/EBNA-1 cells together with a prokaryotic backbone plasmid (Backbone). At the indicated times posttransfection, plasmid DNA was isolated by Hirt extraction and digested with XhoI and DpnI, and the level of replicated, DpnI-resistant DNA was determined by quantitative competitive PCR. PCRs were performed using 105 cell equivalents and a competitor DNA standard curve (9 pg, 3 pg, 600 fg, 120 fg, and 24 fg), except for the day 9 time point (9d.) for the FR experiment, in which 9 pg of competitor was not used. Numbers below each gel refer to the average number of molecules present per transfected cell for each test plasmid (oriP, DS, and FR). The amount of replicated oriP plasmid present at 6 days posttransfection and the amount of replicated DS plasmid present at each time point were quantified from separate gels in which 3.3 × 103 cell equivalents were assayed. The amount of replicated backbone plasmid detected at 6 days posttransfection in the oriP, DS, and FR experiments was 3.0, 3.6, and 0.8 molecules per transfected cell, respectively, and was undetectable at the latter times (<0.2 molecule/transfected cell). The 16-day time point for the oriP test plasmid is not shown because the cells were lost to contamination after the day 13 harvest. (B) DS plasmids are stable during 24 days posttransfection in 293/EBNA-1 cells, while oriP plasmids are lost precipitously. Shown is a graphic representation of experiments conducted as described above. The replication level of each plasmid was plotted versus the days posttransfection. For each independent experiment, the level of replicated test plasmid detected at 6 days posttransfection was set to 100% and the replication level at later time points was set relative to this point (100% = 439 ± 251 oriP and 754 ± 324 DS molecules per transfected cell). The data points are representative of the number of independent experiments listed: 6 days, n = 7; 9 days, n = 3; 12 days, n = 4; 13 days, n = 3; 16 days, n = 2; 18 days, n = 4; 24 days, n = 4. Three experiments in which the replication of the oriP test plasmid was monitored at 6, 9, 13, and 16 days posttransfection were presented previously (37). Note that the level of replicated oriP plasmid decreased approximately 100-fold from the cell population between 6 and 18 days posttransfection, then remained stable between 18 and 24 days. This finding is consistent with our previous observation that oriP plasmids are established in only 1% of transfected 293 cells (37). The 4% rate-of-loss curve, depicted by black cross-hatched boxes, is theoretical and is based on previous studies of established, drug-resistant cell clones (29, 56).

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Replicated DS plasmids are stable during 16 days posttransfection in the 293/1728#5 cell clone, as seen in the 293/EBNA-1 cell clone. The 293/1728#5 cell clone was isolated as described (37). This cell line was selected to stably replicate the 1728 plasmid, which contains oriP and an EBNA-1 expression cassette. The levels of replicated DS plasmid (DS) and prokaryotic backbone plasmid (Backbone) were monitored over 16 days posttransfection as described in the legend to Fig. 3A. Numbers below each gel refer to the average number of replicated DS plasmids present per transfected cell. The replicated backbone plasmid was present at less than one copy per transfected cell for each time point. These data were confirmed by Southern blot analysis (data not shown).

FR of oriP does not limit DNA synthesis at the DS. Why does a plasmid containing only the DS of oriP support stable replication within 293/EBNA-1 cells, yet addition of FR to this plasmid promotes the replicon's precipitious loss from the majority of transfected cells? This observation is counterintuitive, because the FR has been shown to act in cis to promote the maintenance of plasmids containing a putative viral or mammalian origin in 293 and 143B cell lines (30, 34). Given that the DS plasmid supported replication more efficiently than the oriP plasmid at 6 days posttransfection (Wilcoxon rank sum test: P [two-sided] = 0.009; n = 7), we postulated that the FR may impose licensing on the DS of oriP so that it is synthesized only once per cell cycle. In the absence of the FR, the DS plasmid would be amplified each cell cycle, as is the replicon of bovine papillomavirus (BPV), and these DS replicons would be maintained if four EBNA-1-binding sites within the DS are sufficient for plasmid maintenance in 293/EBNA-1 cells.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Plasmid containing only the DS of oriP is synthesized once per cell cycle, as is an oriP replicon (1, 61). Equimolar amounts of an oriP plasmid (oriP) and a plasmid containing only the DS of oriP (DS) were introduced independently into 293/EBNA-1 cells, and the cells were labeled with bromodeoxyuridine for 17 h at 5 days posttransfection. Plasmid DNA was isolated by Hirt extraction of approximately 5 × 107 cells, linearized with XhoI, and separated on a CsCl density gradient. The refractive index of every fourth fraction was determined, one-fourth of each fraction was transferred to a nylon membrane, and plasmid DNA present in the LL, HL, and HH peaks was determined by Southern blotting (48). The relative DNA concentration was plotted versus fraction number. No plasmid DNA was detected at the density at which HH DNAs should migrate. The limit of detection was approximately 1 molecule per transfected cell (25 pg).

Efficient transcriptional activation from an oriP replicon does not promote its precipitous loss. The FR clearly inhibits the establishment of an oriP replicon in 293/EBNA-1 cells, and an epigenetic event is required to overcome this inhibitory activity (37). But how does the FR inhibit establishment? The FR possesses two features that are not shared by the DS. First, the FR acts as a potent transcriptional enhancer, activating transcription of a promoter present in cis by 10- to 100-fold when EBNA-1 is provided in trans (4, 35, 46, 57). Second, when EBNA-1 dimers are bound to the 20 binding sites in the FR, the DNA synthesis machinery cannot proceed efficiently through this region (11, 15). The FR therefore scores as a replication fork barrier (RFB) in two-dimensional gel electrophoresis studies (15). We examined whether either of these attributes of the FR underlies its inhibitory activity.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Efficient transcriptional activation from an oriP plasmid does not promote its precipitous loss. The level of replicated oriP test plasmid (oriP) and oriP test plasmid lacking the thymidine kinase promoter (TKp) was monitored over 16 days posttransfection in 293/EBNA-1 cells as described in the legend to Fig. 3A. The replication level of these plasmids was plotted versus the days posttransfection. For each independent experiment, the level of replicated test plasmid detected at 6 days posttransfection was set to 100%, and the replication level at later time points was set relative to this point (100% = 403 oriP and 318 ± 175 TKp molecules per transfected cell). The loss of the TKp plasmid is representative of three independent experiments conducted in parallel to the oriP test plasmid. The 4% rate-of-loss curve, depicted by black cross-hatched boxes, is theoretical and is based on previous studies of established, drug-resistant cell clones (29, 56).

Multiple EBNA-1 binding sites in FR of oriP promote inefficient establishment of plasmid replicon. Given that the FR's enhancement of transcription from the oriP replicon does not underlie the replicon's precipitous loss, we addressed whether the FR, by acting as a potent RFB, promotes this inefficient establishment. oriP plasmids that initiate DNA synthesis during the last 3 h of S-phase may not be completely synthesized by the end of S-phase, as 3 or more hours are devoted to DNA synthesis through the FR (given that the FR is detected as an RFB in EBV and assuming that DNA elongation occurs at a rate of 1 kbp/min) (15). These plasmids may be eliminated from the cell if a checkpoint is not imposed to ensure duplication of the oriP plasmid prior to entry into G₂ phase. The FR, by acting as an RFB, may thereby promote the loss of plasmids whose origin fires late during S-phase.

View larger version (20K): [in this window] [in a new window]   FIG. 7.   (A) Multiple EBNA-1-binding sites within the FR of oriP inhibit the replicon's establishment. oriP plasmid derivatives containing permutations in the number and type (high versus low affinity) of EBNA-1-binding sites were introduced into 293/EBNA-1 cells, and the level of replicated plasmid DNA was monitored over 24 days posttransfection as described in the legend to Fig. 3A. The oriP and DS plasmids were analyzed in four independent experiments conducted in parallel to the oriP plasmid derivatives shown. (These data are also presented in Fig. 7B and 8, as all oriP plasmid derivatives were analyzed simultaneously.) The replication level of these plasmids was plotted versus the days posttransfection. For each of four independent experiments, the level of replicated test plasmid detected at 6 days posttransfection was set to 100%, and the replication level at later time points was set relative to this point (100% = 625 ± 121 oriP, 967 ± 258 DS, 876 ± 175 9FR+DS, 1149 ± 405 3FR+DS, and 1,498 ± 487 DS+DS molecules per transfected cell). The 3FR+DS plasmid data are representative of three independent experiments. The 4% rate-of-loss curve, depicted by black cross-hatched boxes, is theoretical and is based on previous studies of established, drug-resistant cell clones (29, 56). (B) An RFB from the rDNA locus, when introduced in place of the FR of oriP, does not inhibit establishment of the replicon. oriP plasmid derivatives containing one (RFBDS) or two (RFBDSRFB) copies of the rDNA RFB (16) in place of the FR of oriP were introduced into 293/EBNA-1 cells, and the level of replicated plasmid DNA was monitored over 24 days posttransfection as described in the legend to Fig. 3A. The oriP and DS plasmids were analyzed in four independent experiments conducted in parallel to the oriP plasmid derivatives shown. (The data are also presented in Fig. 7A and 8, as all oriP plasmid derivatives were analyzed simultaneously.) The replication level of these plasmids was plotted versus the days posttransfection. For each of four independent experiments, the level of replicated test plasmid detected at 6 days posttransfection was set to 100%, and the replication level at later times was set relative to this point (100% = 625 ± 121 oriP, 967 ± 258 DS, 1,043 ± 462 RFBDS, and 843 ± 215 RFBDSRFB molecules per transfected cell). The 4% rate-of-loss curve, depicted by black cross-hatched boxes, is theoretical and is based on previous studies of established, drug-resistant cell clones (29, 56).

Spacing between FR and DS does not affect establishment of an oriP replicon. EBNA-1 dimers bound to the FR can associate with EBNA-1 dimers bound to the DS, resulting in the formation of intramolecular "looped" DNA complexes and intermolecular "linked" DNA complexes, as visualized by electron microscopy (14, 54). DNA looping and linking between the FR and DS are not required for replication in that, in 293/EBNA-1 cells, plasmids containing only the DS of oriP are established efficiently. We wished to address whether intramolecular DNA looping between the FR and DS could contribute to the inefficient establishment of oriP replicons. Given that it is less energetically favorable for a trans-acting factor to loop two cis elements when the distance between those elements is decreased (20), we constructed an oriP plasmid derivative in which the 963 bp separating FR and DS were deleted (FR*DS) (Fig. 8). DNA looping is unlikely to occur in the FR*DS plasmid, as Su et al. did not detect looped DNA complexes in electron microscopy studies with a DNA fragment containing the FR, and the length of the loops with an oriP DNA fragment detected ranged from 1,000 to 1,780 bp (54). DNA loops between the juxtaposed FR and DS would be less than 800 bp.

View larger version (18K): [in this window] [in a new window]   FIG. 8.   Spacing between the FR and DS of oriP has no effect on establishment of the replicon. The 963 nucleotides separating the FR and DS of oriP were deleted in the context of the oriP test plasmid and the 3FR+DS plasmid, the resulting plasmids (FR*DS and 3FR*DS, respectively) were introduced into 293/EBNA-1 cells, and the level of replicated DNA was monitored over 24 days posttransfection as described in the legend to Fig. 3A. The oriP and DS plasmids were analyzed in four independent experiments conducted in parallel to the oriP plasmid derivatives shown. (The data are also presented in Fig. 7A and 7B, as all oriP plasmid derivatives were analyzed simultaneously.) The replication level of these plasmids was plotted versus the days posttransfection. For each of four independent experiments, the level of replicated test plasmid detected at 6 days posttransfection was set to 100%, and the replication level at later times was set relative to this point (100% = 625 ± 121 oriP, 967 ± 258 DS, 439 ± 222 FR*DS, and 817 ± 576 3FR*DS molecules per transfected cell). The 3FR*DS data are representative of three independent experiments except for the day 24 time point, which is representative of two experiments. The 4% rate-of-loss curve, depicted by black cross-hatched boxes, is theoretical and is based on previous studies of established, drug-resistant cell clones (29, 56).

	DISCUSSION

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Cell lines vary in their ability to support replication of oriP plasmid derivatives. We have monitored the fate of plasmids containing oriP or only the DS of oriP for 24 days following their introduction into various EBNA-1-positive cell lines. While oriP plasmids are lost precipitously and established inefficiently in all 22 cell lines analyzed to date (27, 37, 45, 58), surprisingly we have found that cell lines vary dramatically in their ability to support the replication of a DS plasmid. DS plasmids were eliminated rapidly from 143B cells, so that the level of replicated DS plasmid detected at 4 days posttransfection was only 10% of the level of wild-type oriP and dropped below the level of detection (<0.1 molecule/cell) by 7 days posttransfection. Replicated DS plasmids were lost at an intermediate rate in C33A/EBNA-1 cells. That is, the DS plasmid supported replication as efficiently as the oriP plasmid at 6 days posttransfection (Wilcoxon rank sum test: P [two-sided] = 0.28; n = 3) and was lost at a rate of approximately 28% per cell generation between 6 and 16 days following transfection, as was the oriP plasmid. In 293/EBNA-1 cells, however, the DS plasmid was established efficiently, being lost at a rate of only 8% per cell generation, while the oriP plasmid was lost at a rate of 33% per cell generation between 6 and 18 days posttransfection. This replicated DS plasmid was likewise stable in the 293/1728#5 cell clone.

FR can stimulate and inhibit replication of oriP replicons. EBNA-1 binding sites from the FR clearly contribute to DNA synthesis and/or maintenance of oriP plasmids in some cell lines. That is, we and others have shown that oriP plasmid derivatives containing fewer than seven EBNA-1 binding sites from the FR fail to support short-term replication in Raji, D98/Raji, and EBNA-1-positive 143B cells (8, 30, 47, 57). However, even with the intact FR, oriP plasmids are established in only 1 to 10% of transfected cells (37). By monitoring the replication of oriP plasmid derivatives in multiple cell lines, we unexpectedly found that the FR also acts in cis to promote the rapid loss of replicated oriP plasmids from the population of transfected 293/EBNA-1 cells and 293/1728#5 cells. This inhibitory activity of the FR likely underlies the inefficient establishment of oriP replicons in all cells, as oriP replicons are established inefficiently in all 21 cell lines analyzed to date (27, 37, 45, 58). Previously we have shown that oriP plasmids must be modified epigenetically for establishment (37). (Note that in Fig. 3B the level of replicated oriP plasmid decreased approximately 100-fold from the cell population between 6 and 18 days posttransfection, then remained stable between 18 and 24 days. This is consistent with our previous observation that oriP plasmids are established in only 1% of transfected 293 cells [37].) Our findings indicate that this epigenetic event is required to overcome an inhibitory activity of the FR.