The Linking Regions of EBNA1 Are Essential for Its Support of Replication and Transcription

Plasmids.

The EBNA1 expression plasmids p1160 and p1553, which contain the genes 1160 and 1553, respectively, have been described previously (2, 45). Standard PCR-based cloning strategies were used to generate the new derivatives. The expression of all the derivatives of EBNA1 is under the control of the cytomegalovirus (CMV) immediate-early (IE) promoter. The derivatives contain only amino acids from EBNA1, with the following exceptions: 1893 has a valine and a methionine following the Gly-Gly-Ala repeats and preceding amino acid 378, 1767 has a leucine and a methionine following the Gly-Gly-Ala repeats, 1763 has an alanine and a methionine following the Gly-Gly-Ala repeats, 1891 has a valine and a methionine preceding amino acid 378, and 1747 has a serine and a methionine following the Gly-Gly-Ala repeats. An ABI automated DNA sequencer (Applied Biosystems) was used to verify the sequence of each derivative. pCDNA3 (Invitrogen), which has the CMV IE promoter but expresses no protein, served as a negative control.

The replication reporter plasmids were 1591 (with oriP) and 1381 (without oriP). Plasmid 1381 has been described previously (32). Plasmid 1591 was constructed as follows. The AatII-to-BamHI fragment from 994 (31), which contains oriP, was introduced into the AatII and BamHI sites in pUC19. The resulting plasmid was then cut with StuI and HindIII (the HindIII site was blunted with the Klenow fragment), and a BsmBI-to-BamHI fragment (each site was blunted with the Klenow fragment), which contains the simian virus 40 (SV40) IE promoter driving the expression of the neomycin resistance gene, was introduced. The insert came from a derivative of pSV2Neo (56) which had previously been cut with NsiI, blunted with mung bean nuclease, and religated. This fragment, which contains only one of the 72-bp repeats from SV40, is oriented with the promoter from SV40 driving transcription away from the DS of oriP.

The transcription reporter plasmid FR-TK-Luciferase has been described previously (44). Briefly, this plasmid contains the FR of oriP upstream of the herpes simplex virus thymidine kinase promoter driving the expression of the luciferase gene. In the transcription assays, pEQ176 (52), which has the CMV IE promoter driving the expression of the β-galactosidase gene, was used as an internal control for transfection efficiency.

Cells and transfections.

143B (7) and 293 (23) cells were grown in Dulbecco modified Eagle medium supplemented with 10% bovine calf serum and fetal calf serum, respectively. Plasmids were transfected into both cell types as a calcium phosphate precipitate (51). The precipitate was placed on adherent 293 cells in a standard fashion. 143B cells were suspended by trypsinization, combined with the precipitate, and placed in a new tissue culture dish. For each cell type, the medium was refreshed 6 to 8 h after the addition of the precipitate.

Western blotting.

Between 1 and 6.4 μg of each EBNA1 expression plasmid was transfected into 293 cells. We adjusted the amount of each expression plasmid that was transfected in order to normalize the expression of the derivatives. The total amount of CMV IE promoter-containing plasmid used per transfection was kept constant by supplementation with pCDNA3. The cells were grown for 48 h prior to harvest. Western blotting was done by standard protocols (51). Briefly, extracts were separated on sodium dodecyl sulfate–10% polyacrylamide gels and transferred to nitrocellulose membranes. After blocking was done with 10% fat-free milk and 0.05% Tween 20, blots were exposed to a rabbit anti-EBNA1 polyclonal antiserum. This antiserum was raised against a fusion protein containing amino acids 7 to 37 and 420 to 617 of EBNA1 and primarily or entirely recognizes the DNA-binding domain (57). The secondary antibodies were either alkaline phosphatase-conjugated goat anti-rabbit antibodies (Boehringer Mannheim Biochemicals) (data not shown) or ³⁵S-labeled donkey anti-rabbit antibodies (Amersham) (Fig. 1B). The ³⁵S-labeled blot was visualized and quantified with a PhosphorImager (Molecular Dynamics).

Replication assays.

143B cells were transfected with 1 to 6.4 μg of each EBNA1 expression plasmid, 10 μg of 1591, and 7.3 μg of 1381 (equimolar amounts of 1591 and 1381). After 96 h, the cells were harvested, and low-molecular-weight DNA was extracted by the method of Hirt (28) and digested exhaustively with DpnI. A modified version of a quantitative competitive PCR assay (32) was used to quantify the amounts of DpnI-resistant 1591 and 1381; the modified assay did not use radioactive primers. The PCR products of 1591, 1381, and a competitor were resolved in an agarose gel, visualized with ethidium bromide, and quantified with a charge-coupled device camera (IS-1000 digital imaging system; Alpha Innotech Corporation). The amounts of 1591 and 1381 were determined by identifying the amount of competitor DNA which yielded the same amount of product.

Transcription assays.

Cells were transfected with 0.1 to 0.64 μg of each EBNA1 expression plasmid (10% as much of each derivative as was used for Western blotting and replication assays), 100 ng of FR-TK-Luciferase, and 100 ng of pEQ176 (see above). Cells were grown for 48 h and harvested, and luciferase activity and β-galactosidase activity were measured (with kits from Bio-Rad and Tropix, respectively). The β-galactosidase activity was used to normalize the luciferase activity from each transfection.

DNA-linking assays.

293 cells were transfected with 1 to 6.4 μg of each EBNA1 expression plasmid, and whole-cell extracts were prepared. Cell pellets were resuspended at 10⁸ cells/ml in a buffer consisting of 50 mM Tris (pH 7.5), 5 mM EDTA, 0.8 M NaCl, and 0.3% Nonidet P-40, sonicated, and incubated at room temperature for 15 min, and then an equal volume of 50 mM Tris (pH 7.5) was added. The extracts were microcentrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was used. A similar extract was prepared from untransfected 293 cells. The total amount of extract in each reaction was kept constant. The probe DNA, which is 131 bp long and contains two binding sites for EBNA1, is the SalI-to-PstI fragment from p880 (45). This fragment was purified from an agarose gel, quantified, and labeled by filling in the ends with the Klenow fragment in the presence of [α-³²P]dCTP (Amersham). Extracts, 4 fmol of probe, and 1.35 μg of poly(dl) · poly(dC) (Pharmacia) were combined in reaction mixtures with a final NaCl concentration of 0.3 M and incubated at room temperature for 30 min. Reactions were resolved by electrophoresis at 15 to 25 mA through 4% polyacrylamide gels in 0.5× TBE (45 mM Tris-borate, 1 mM EDTA). Gels were dried under vacuum at 80°C onto 3MM chromatography paper (Whatman), and a PhosphorImager was used to visualize and quantify the data.

Structures of derivatives of EBNA1.

We wished to determine whether DNA linking contributes to EBNA1 activities in cells. Because the linking regions are large and contain multiple domains which can independently mediate linking (39), subtle mutations which substantially affect linking are unlikely to be identified readily. Therefore, we constructed derivatives of EBNA1 with gross deletions or rearrangements of the linking regions (Fig. 1A). Derivatives of EBNA1 in one set each contain two linking regions. These derivatives contain either linking region 1 and linking region 2 in various contexts (1553, 1743, and 1745) or substitutions of linking region 1 for linking region 2 (1893) and of linking region 2 for linking region 1 (1767). Derivatives in a second set contain one linking region in various contexts (1708, 1710, 1763, 1891, and 1747). A final set consists of derivatives containing neither linking region (1709 and 1160). All of the derivatives contain the nuclear localization sequence within amino acids 379 to 391 (5). Each derivative also contains amino acids 451 to 641, which include the necessary residues for dimerization and site-specific DNA binding (5, 10, 12, 29). Several derivatives contain a truncated, 15-amino-acid version of the Gly-Gly-Ala repeats. Aside from the truncated Gly-Gly-Ala repeats, the 1553 derivative is identical in sequence to EBNA1 from the B95-8 strain of EBV (8). The 1553 derivative and EBNA1 from B95-8 indistinguishably support replication and transcription in tissue cultures (69); therefore, the 1553 derivative is considered wild type in this study.

Each derivative of EBNA1 was detectably expressed following its transfection into 293 cells (Fig. 1B). The amounts of DNA transfected were chosen to minimize both the range of expression of derivatives and the range in levels of DNA needed to provide this expression. From 1 to 6.4 μg of each DNA was transfected; these amounts provided levels of expression of each derivative within 10-fold of one another. The variation in both the amount of DNA transfected and the level of the derivatives expressed is independent of their activities (compare Fig. 1B, 2, and 3). The specific amount of each plasmid used in this calibration experiment was identical to the amounts used in the replication assays, and 1/10 as much of each plasmid was used in the transcription assays.

Ability of derivatives of EBNA1 to support replication.

We determined the ability of derivatives of EBNA1 to support replication in a short-term assay (Fig. 2A). Expression plasmids for each derivative of EBNA1 were cotransfected with a replication reporter plasmid (containing oriP) and a replication control plasmid (lacking oriP) into 143B cells. After 96 h, the cells were harvested and low-molecular-weight DNAs were isolated by Hirt extraction (28). The transfected DNA was prepared from dam⁺ Escherichia coli and thus was sensitive to cleavage by DpnI. DpnI cleaves DNA that is fully methylated or hemimethylated by dam methylase (3); therefore, only plasmids which have undergone two rounds of synthesis will yield DNA resistant to digestion with DpnI. A quantitative competitive PCR assay (32) was used to measure the extent of replication of the plasmids containing and lacking oriP. This assay has been validated previously by use of Southern blotting with related oriP plasmids in the same cells (32).

The ability of each derivative of EBNA1 to support the replication of an oriP plasmid in this short-term assay was measured in four independent experiments (Fig. 2B). Within each experiment, the amount of replicated oriP plasmid in cells expressing 1553 was set to 100%. In general, derivatives with two linking regions supported replication in a manner similar to that of 1553, derivatives with one linking region supported a reduced level of replication, and derivatives with neither linking region did not support replication above the background level observed with pCDNA3 (control plasmid expressing no EBNA1). Two exceptions to this generalization are 1745 and 1710, which are similar to 1743 and 1708, respectively, but lack amino acids 392 to 450 and are impaired in their support of replication. Thus, to support oriP-dependent replication fully, EBNA1 requires two linking regions and amino acids 392 to 450. In a parallel experiment, derivatives of EBNA1 supported the replication of oriP similarly in 293 cells and in 143 cells. Derivatives 1893 and 1767, each with two linking regions, had activities similar to that of 1553; derivatives 1891 and 1708, with only one linking region, had reduced activities; and derivative 1160, lacking linking regions, had activity close to background activity (data not shown).

Linking region 1 and linking region 2 are equivalent in their contributions to the support of replication by derivatives of EBNA1. Derivatives with either linking region alone, excluding 1710, which lacks amino acids 392 to 450, supported replication similarly (10 to 23% as efficiently as 1553). Furthermore, derivatives in which each linking region was substituted for the other (1893 and 1767) supported replication as well as did 1553. That linking region 1 and linking region 2 contribute equally to the support of replication by EBNA1 indicates that linking per se or some other activity shared between them contributes to the support of DNA replication by EBNA1.

Ability of derivatives of EBNA1 to support transcription.

Expression plasmids for each derivative of EBNA1 were cotransfected with FR-TK-Luciferase (Fig. 3A) into 143B (Fig. 3B) or 293 (Fig. 3C) cells. Cells were harvested 48 h after transfection, and luciferase activity was measured. Within each experiment, the luciferase activity in cells expressing 1553 was set to 100%. The average signals with the control plasmid, pCDNA3, were 1.3% in 143B cells and 7.9% in 293 cells.

The linking regions are not equivalent in their support of transcription. In 143B cells (Fig. 3B), the derivative containing two copies of linking region 2 (1767), which fully supported oriP-dependent replication, was significantly impaired in its support of transcription. Otherwise, the ability of the derivatives to support transcription mirrored their ability to support replication. Derivatives which contained only one linking region (1708, 1710, 1763, 1891, and 1747) were much less active than derivatives with both linking regions (1553 and 1743) or two copies of linking region 1 (1893) but were more active than derivatives with neither linking region. Application of the Wilcoxon rank sum test indicated that, in 143B cells, each derivative with one linking region supported transcription significantly better than 1709 and 1166 (P, <0.05), except for 1763 (P, 0.09). The ability of derivatives of EBNA1 to support transcription in 293 cells (Fig. 3C) differed from that in 143B cells. In 293 cells, derivatives containing one copy of linking region 2 (1708, 1710, and 1763) supported transcription in a manner comparable to that of 1553, and duplication of linking region 2 (1767) did not augment this activity. In 293 cells, as in 143B cells, derivatives containing one copy of linking region 1 (1891 and 1747) supported transcription slightly, although significantly better than 1709 and 1160 (P, <0.03), and the derivative with a duplication of linking region 1 (1893) supported transcription fully. The contribution of linking region 1 but not that of linking region 2 to the support of transcription by EBNA1 can be augmented by duplicating that region within EBNA1.

The deletion of amino acids 392 to 450 diminished the ability of EBNA1 to support transcription. In each cell type, 1745 and 1710 were less active than 1743 and 1708, respectively. However, this deletion decreased the support of transcription less markedly than it affected the support of replication.

Ability of derivatives of EBNA1 to link DNA.

The ability of derivatives of EBNA1 to link DNA was determined with an electrophoretic mobility shift assay (39). In this assay, DNAs linked by EBNA1 are incorporated into large complexes which are unable to migrate appreciably into the gel. This assay has been validated previously by electron microscopic analyses of intramolecular linking (19, 59) and by enhancement of ligation of DNAs mediated by their linkage (39). A representative experiment for a subset of the derivatives is shown in Fig. 4A. Various amounts of whole-cell extract expressing derivatives of EBNA1 were incubated with a DNA containing two binding sites for EBNA1 in the presence of a constant, total amount of cell extract. The amounts of EBNA1-expressing extract were adjusted so that the percentage of bound DNA spanned 50% for each derivative. For each derivative tested, the percentage of DNA linked was plotted against the percentage bound (Fig. 4B). These plots were used to interpolate the percentage of DNA linked by each derivative when 50% of the DNA was bound, and this value was defined as the linking activity of that derivative.

The linking activity of derivatives of EBNA1 is a function of the number of linking regions that they contain. The linking assay was conducted three times (Fig. 4C), and in each experiment the linking activity of 1553 was set to 100%. In all three experiments, the derivatives with two linking regions (1553, 1743, 1893, and 1767) had the highest linking activities, the derivatives with one linking region (1708, 1763, 1891, and 1747) had intermediate linking activities, and the derivatives with neither linking region (1709 and 1160) had the lowest linking activities. Derivatives of EBNA1 with one linking region link DNAs more efficiently than those with neither linking region but less efficiently than those with two linking regions.

DNA linking correlates with biological activities.

The linking activity of derivatives of EBNA1 measured in Fig. 4 was compared to their activities measured in cells in Fig. 2 and 3. Because amino acids 392 to 450 do not contribute to DNA linking (39) but do affect the support of replication (Fig. 2) and transcription (Fig. 3) by EBNA1, derivatives lacking them (1710 and 1745) are not included in these comparisons (Fig. 5). For the derivatives of EBNA1 tested in Fig. 4, their average linking activity was plotted against their support of oriP-dependent replication (Fig. 5A) and transcription in 143B (Fig. 5B) and 293 (Fig. 5C) cells. The application of the Kendall rank correlation test indicated that linking activity correlates with the support of replication (P, 0.003) and transcription in 143B (P, 0.003) and 293 (P, 0.02) cells. These correlations are consistent with DNA linking contributing to the support of replication and transcription by EBNA1.