The Minimal Replicator of Epstein-Barr Virus oriP

Abstract

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports the replication and stable maintenance of plasmids in human cells. oriP contains two essential components, called the DS and the FR, both of which contain multiple binding sites for the EBV-encoded protein, EBNA-1. The DS appears to function as the replicator of oriP, while the FR acts in conjunction with EBNA-1 to prevent the loss of plasmids from proliferating cells. Because of EBNA-1's role in stabilizing plasmids through the FR, it has not been entirely clear to what extent EBNA-1 might be required for replication from oriP per se, and a recent study has questioned whether EBNA-1 has any direct role in replication. In the present study we found that plasmids carrying oriP required EBNA-1 to replicate efficiently even when assayed only 2 days after plasmids were introduced into the cell lines 143B and 293. Significantly, using 293 cells it was demonstrated that the plasmid-retention function of EBNA-1 and the FR did not contribute significantly to the accumulation of replicated plasmids, and the DS supported efficient EBNA-1-dependent replication in the absence of the FR. The DS contains two pairs of closely spaced EBNA-1 binding sites, and a previous study had shown that both sites within either pair are required for activity. However, it was unclear from previous work what additional sequences within the DS might be required. We found that each "half" of the DS, including a pair of closely spaced EBNA-1 binding sites, had significant replicator activity when the other half had been deleted. The only significant DNA sequences that the two halves of the DS share in common, other than EBNA-1 binding sites, is a 9-bp sequence that is present twice in the "left half" and once in the "right half." These nonamer repeats, while not essential for activity, contributed significantly to the activity of each half of the DS. Two thymines occur at unique positions within EBNA-1 binding sites 1 and 4 at the DS and become sensitive to oxidation by permanganate when EBNA-1 binds, but mutation of each to the consensus base, adenine, actually improved the activity of each half of the DS slightly. In conclusion, the DS of oriP is an EBNA-1-dependent replicator, and its minimal active core appears to be simply two properly spaced EBNA-1 binding sites.

The plasmid replicon of Epstein–Barr virus: Mechanistic insights into efficient, licensed, extrachromosomal replication in human cells

“…While the virus has evolved to maintain 20 EBNA1-binding sites in FR, efficient plasmid maintenance requires only 7 of these sites, and can be reconstituted by a polymer of 3 copies of DS, having a total of 12 EBNA1-binding sites (Wysokenski and Yates 1989). The Dyad Symmetry element (DS) is sufficient for initiation of DNA synthesis in the presence of EBNA1 (Aiyar, Tyree et al 1998), (Yates, Camiolo et al 2000), and initiation occurs either at or near DS (Gahn and Schildkraut 1989); (Niller, Glaser et al 1995). Termination of viral DNA synthesis is thought to occur at FR, because when FR is bound by EBNA1 it functions as a replication fork barrier as observed by 2D gel electrophoresis (Gahn and Schildkraut 1989), (Ermakova, Frappier et al 1996), (Wang, Lindner et al 2006).…”

“…One protein found to bind these elements, TRF2, exhibits cooperative binding with a truncated form of EBNA1 at DS as measured in gel shift assays, and may contribute to the enhanced replication of DNA by increasing the apparent affinity of EBNA1 for its binding sites (Deng, Lezina et al 2002). Despite this two-fold effect, the nonamer elements are not necessary for replicative function (Julien, Polonskaya et al 2004), (Yates, Camiolo et al 2000), (Koons, Van Scoy et al 2001), (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003;Wang, Lindner et al 2006). Genetic dissections of DS indicate that the minimal requirement for its replicative function is one pair of EBNA1-binding sites (Harrison, Fisenne et al 1994), (Shirakata and Hirai 1998), (Yates, Camiolo et al 2000), (Koons, Van Scoy et al 2001), (Bashaw and Yates 2001).…”

“…Despite this two-fold effect, the nonamer elements are not necessary for replicative function (Julien, Polonskaya et al 2004), (Yates, Camiolo et al 2000), (Koons, Van Scoy et al 2001), (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003;Wang, Lindner et al 2006). Genetic dissections of DS indicate that the minimal requirement for its replicative function is one pair of EBNA1-binding sites (Harrison, Fisenne et al 1994), (Shirakata and Hirai 1998), (Yates, Camiolo et al 2000), (Koons, Van Scoy et al 2001), (Bashaw and Yates 2001). The spacing between a pair of EBNA1-binding sites is critical as well; changing the center-to-center spacing of the sites by inserting or deleting only 1bp eliminates DNA synthesis (Bashaw and Yates 2001).…”

“…Insertions of 3, 5, or 10bp between the paired sites do not support DNA replication either (Harrison, Fisenne et al 1994). Additionally, pairs of EBNA1-binding sites within the EBV genome that have center-to-center spacings other than 21bp, such as in FR (with 30bp spacing) and near the Q promoter (with 24bp spacing) (Figure 1.A), do not support DNA replication (Reisman, Yates et al 1985), (Aiyar, Tyree et al 1998), (Yates, Camiolo et al 2000). The functional roles of the elements within DS have been confirmed by studies of another region of EBV's genome, termed Rep*, which was identified as an element that can substitute for DS inefficiently (Kirchmaier and Sugden 1998).…”

“…Additionally, there is a region of helically unstable DNA near DS that has been proposed to facilitate DNA unwinding and the initiation of DNA (Polonskaya, Benham et al 2004). Flanking the pairs of EBNA1-binding sites of DS are three 9bp elements that resemble telomeric repeats, and have been termed nonamers (Niller, Glaser et al 1995), (Yates, Camiolo et al 2000). Lieberman and colleagues have shown in several studies that these elements are bound by cellular proteins associated with the telomeres, including TTAGGG-Repeat binding Factors (TRF1, TRF2), the human repressor activator protein 1 (hRap1), and tankyrase (Deng, Lezina et al 2002), (Deng, Atanasiu et al 2003), (Deng, Atanasiu et al 2005).…”