The Minimal Replicator of Epstein-Barr VirusoriP

Dependence of oriP-specific plasmid replication on EBNA-1 in 143B cells during the first 48 h after transfection. Duplicate dishes of 143B cells were transfected with test plasmids, pHyg (vector, “−”), pHEBo (pHyg carryingoriP, “wt”), or pHEBo carrying different mutations inoriP, as indicated for samples 1 to 10. For samples 11 and 12, cells were transfected with p367, which is pHEBo carrying the EBNA-1 gene. Plasmids were harvested from cells 48 h after transfection. For Southern analysis, 40% of each sample was digested with BamHI plus DpnI (upper blot), and 10% was digested with BamHI alone (lower blot). The percentages of total plasmid (lower blot) that replicated (full-length,DpnI-resistant plasmid, upper blot) were calculated by comparison to the quantitative standards of each blot. To test for the completeness of DpnI cutting, 2 ng of pHEBo was combined with DNA extracted from mock-transfected cells and digested withDpnI plus BamHI (lane marked “Co.,” upper blot). The standards were linearized pHEBo: 125 and 32 pg for the upper gel and 500 and 125 pg for the lower gel.

The DS component of oriP is an EBNA-1-dependent replicator. Duplicate plates of 293 cells were transfected with the oriP test plasmids as indicated. Cells were also cotransfected with either p367, a derivative of pHEBo that produces EBNA-1 (lanes 10 to 17), or with p396, a similar plasmid that produces mutant EBNA-1 that cannot bind to DNA (lanes 2 to 9). Plasmids were extracted from cells 46 h after transfection and were analyzed for DpnI-resistant plasmids (40% of each sample, upper blot) and total plasmids (2% of each sample, lower blot). The blots were processed in parallel using the same labeled probe, and signals from beta-image analysis were compared directly to calculate the percentages of replicated DNA. Stimulation by EBNA-1 is the ratio of the average amount of replication of each test plasmid in the presence of EBNA-1 to that in the absence of EBNA-1. NA, not applicable. A control for DpnI digestion was included in lane 1, upper blot, as was done for Fig. 2. The lower part of the gel containing the products of DpnI digestion is not shown.

Analysis of EBNA-1 binding to DS mutants lacking individual binding sites, as determined by EMSA. An end-labeled, 278-bp DNA including the DS was prepared by PCR from wild-type oriPor from mutants lacking individual EBNA-1 binding sites, as indicated. EBNA-1 NΔ407 (26) was mixed with each DNA, using a molar ratio of EBNA-1 dimers to DNA of either 4 (for lanes 1, 3, 6, 9, 12, and 15) or 16 (for lanes 4, 7, 10, 13, and 16). EBNA-1 was omitted from lanes 2, 5, 8, 11, and 14. Complexes were electrophoresed through a 4% polyacrylamide gel. At the left, the positions of free probe (F) and complexes with EBNA-1 bound to one site, two sites, three sites, or all four sites are indicated. Complexes with two sites bound have different mobilities, depending primarily on whether the sites are in helical phase (2 IP) or out of helical phase (2 OP) (see the text).

Long-term maintenance of plasmids carrying mutations inoriP in Raji cells. Shown is a Southern analysis of plasmids extracted from 3 million cells of two or four individual hygromycin B-resistant Raji clones carrying pHEBo (WT) or its mutant derivatives, as indicated at the top. The positions of supercoiled (CCC), relaxed circular (RC), and linear (L) forms of pHEBo are indicated at the left. “Chr.” marks the position of residual, fragmented chromosomal DNA, visible on the stained gel. As standards, 200- and 50-pg amounts of pHEBo were loaded in lanes 1 and 22, corresponding to 9 and 2.3 molecules per cell. The numbers of supercoiled monomeric plasmids detected per cell are given as the average for the bands that are bracketed.

Support of long-term plasmid maintenance in 143/98.2 cells by oriP with various mutations at the DS. (A) Duplicate plates of cells were transfected with pHEBo (WT) or its derivatives carrying the indicated mutations at the DS. One-tenth of the cells were selected for hygromycin B resistance and then split successively 1:4 and 1:3 before being expanded and harvested at 18 to 21 days after transfection, except that in the case of the Δ1 3-Bst mutant, cells were not split and required 29 days for expansion. Plasmids extracted from ∼7 million cells were analyzed by Southern blot. For each plasmid tested, the average number of plasmid molecules detected per cell is indicated. The supercoiled (CCC) and relaxed circular (RC) forms of the plasmids are indicated. Note that the vector portion of the plasmids tested in lanes 15, 16, 19, and 20 is slightly smaller than the vector portion of the others (Table 1). (B) To convert the relative amounts detected in panel A to copies per cell, the samples of lanes 1 and 20 in panel A were analyzed again on this blot, in lanes 3 and 4, and compared to known amounts of pHEBo in lanes 1 and 2.

Activities of oriP mutants with substitutions of the nonamer repeats. (A) Duplicate plates of 143/98.2 cells were transfected with pHEBo or its derivatives carrying the indicated mutations. Cultures were split 1:10 before selection for hygromycin resistance, split 1:6 and 1:10 after selection, and harvested 20 to 21 days (lanes 2 to 11) or 24 days (lanes 12 to 23) after transfection. Plasmids extracted from approximately 5 million cells were analyzed by Southern blot. In lane 1, 50 pg of pHEBoΔRV was loaded as a standard corresponding to 1.4 copies per cell. The average calculated numbers of plasmid copies per cell are indicated. (B) Similar experiment to that shown in panel A except that cultures were grown without hygromycin B beyond the 13th day following transfection and the cultures were harvested sooner. After selection, the resistant cultures were split successively 1:10 and then 1:2, except as noted below, and harvested at 15 to 16 days following transfection. With Δ3-4, c-Stu, the cultures could only be split once at 1:5 after selection and before harvesting. With Δ1-4, the cultures were not split after selection and could not be harvested until 24 days posttransfection. The relative average numbers of plasmids detected have been indicated, with pHEBo (WT) set arbitrarily at 10.