Initiation of DNA replication within oriP is dispensable for stable replication of the latent Epstein-Barr virus chromosome after infection of established cell lines

The 165-kb circularized chromosome of Epstein-Barr virus (EBV) is replicated in latently infected cells once per cell cycle by host proteins during S phase. Replication initiates at multiple sites on latent EBV chromosomes, including within a 1.8-kb region called oriP, which can provide both replication and stabilization for recombinant plasmids in the presence of the EBV-encoded protein, EBNA-1. Replication initiates at or near the dyad symmetry component (DS) of oriP, which depends on multiple EBNA-1 binding sites for activity. To test the importance of the replication function of oriP, the DS was deleted from the viral genome. EBV mutants lacking the DS and carrying a selectable gene could establish latent infections in BL30 cells, in which circular, mutant viral chromosomes were stably maintained. Analysis of replication fork movement using two-dimensional gel electrophoresis showed that the deletion of the DS reduced the initiation events to an undetectable level within the oriP region so that this segment was replicated exclusively by forks entering the region from either direction. A significant slowing or stalling of replication forks that occurs normally at the approximate position of the DS was also eliminated by deletion of the DS. The results confirm the DS as both a replication origin and a place where replication forks pause. Since the replication function of oriP is dispensable at least in certain cell lines, the essential role of EBNA-1 for infection of these cell lines is likely to be that of stabilizing the EBV chromosome by associating with the 30-bp repeats of oriP. The results also imply that in established cell lines, the EBV chromosome can be efficiently replicated entirely from origins that are activated by cellular factors. Presumably, initiation of replication at the DS, mediated by EBNA-1, is important for the natural life cycle of EBV, perhaps in establishing latent infections of normal B cells.     

Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein-Barr virus

Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1, 800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.     

The minimal replicator of Epstein-Barr virus oriP

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.     

Plasmid maintenance of derivatives of oriP of Epstein-Barr virus.

oriP is the origin of plasmid replication of Epstein-Barr virus. Replication from oriP requires both the cis-acting elements (the family of repeats and the dyad symmetry element) and the viral origin-binding protein, EBNA-1. The ability of plasmids containing oriP to be maintained stably in EBNA-1-positive cells reflects the efficiency both of their replication and of their segregation each cell cycle. The efficiency of plasmid maintenance was determined for plasmids containing derivatives of oriP with one copy of the dyad symmetry element and two copies of the family of repeats by measuring the rate at which they were lost from cells in the absence of selection. These measurements demonstrated that plasmids with derivatives of oriP with two copies of the family of repeats in one orientation are maintained only slightly less efficiently than is wild-type oriP. To determine whether plasmid maintenance could be affected by reinitiation at the dyad symmetry element (T. A. Gahn and C. L. Schildkraut, Cell 58:527-535, 1989), plasmids containing derivatives of oriP with two copies of the dyad symmetry element and one copy of the family of repeats were compared with plasmids containing wild-type oriP in EBNA-1-positive cells. These measurements showed that plasmids containing a derivative of oriP with two copies of the dyad symmetry element are maintained as efficiently as is wild-type oriP and are not amplified relative to wild-type oriP. These observations indicate that the trans-acting factors that regulate DNA to replicate once per S phase are insensitive to multiple cis-acting regulatory sites within a replicon.     

Single-stranded structures are present within plasmids containing the Epstein-Barr virus latent origin of replication.

The Epstein-Barr virus (EBV) latent origin of plasmid replication (oriP) contains two essential regions, a family of repeats with 20 imperfect copies of a 30-bp sequence and a dyad symmetry element with four similar 30-bp repeats. Each of the repeats has an internal palindromic sequence and can bind EBNA 1, a protein that together with oriP constitutes the only viral element necessary for EBV maintenance and replication. Using single-strand-specific nucleases, we have probed plasmids containing oriP-derived sequences for the presence of secondary structural elements. Multiple single-stranded structures were detected within the oriP region. Of the two essential elements of oriP, the family of repeats seemed to extrude these structures at a much higher frequency than did sequences within the dyad symmetry region. Though negative supercoiling was found to stabilize the single-stranded structures, they showed significant stability even after linearization of the oriP plasmids. Two major single-stranded structures detected involved approximately 12 bp of DNA. These loci could be transiently unwound regions that form because of negative supercoiling and the high A + T content of this region of DNA, or they could be cruciform structures extruded within the palindromic sequences of oriP that may be important sites for protein-DNA interactions in the EBV oriP.     

The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.     

Prolonged gene expression in primary porcine pancreatic cells using an Epstein-Barr virus-based episomal vector

Epstein-Barr virus (EBV)-based plasmids containing the origin of replication (oriP) and EBV nuclear antigen 1 (EBNA-1) are well known for the stable episomal maintenance in human cells. In order to clarify whether an EBV-based plasmid can be maintained stably in the porcine pancreatic cells which are the primary candidate sources of islet xenotransplantation, we constructed pEBVGFP encoding the green fluorescent protein (GFP). Monolayer culture of the porcine neonatal pancreatic cells was lipofected with pEBVGFP or pGFP which was derived from pEBVGFP by deleting out oriP and EBNA-1. pEBVGFP significantly prolonged GFP expression not only in human cell lines but also in the primary porcine pancreatic cells compared with pGFP. Interestingly, the duct cells that are believed as the pancreatic precursor cells were preferentially transfected and conveniently enriched among the mixed primary cell populations using a hygromycin B selection. To our knowledge, this is the first report suggesting the potential application of an EBV-based plasmid for the extended gene expression in the primary porcine pancreatic duct cells.     

Effect of number and position of EBNA-1 binding sites in Epstein-Barr virus oriP on the sites of initiation, barrier formation, and termination of replication.

DNA replication intermediates of three plasmids containing all or part of a modified Epstein-Barr virus cis-acting plasmid maintenance region (oriP) were examined to further investigate oriP function. Replication intermediates were analyzed in vivo and in vitro by neutral-neutral two-dimensional gel electrophoresis. The major functional components of the wild-type oriP are a 140-bp dyad symmetry region (single dyad) and 20 tandem copies of a repeat with a 30-bp consensus sequence (family of repeats). A modified oriP was constructed by replacing the family of repeats with three tandem copies of the single dyad (D. A. Wysokenski and J. L. Yates, J. Virol. 63:2657-2666, 1989). Initiation was observed in vivo near the single dyad in the modified oriP, as seen in the wild-type oriP (T. A. Gahn and C. L. Schildkraut, Cell 58:527-535, 1989), but was not observed near the tandem dyads. A replication barrier and termination were observed near the tandem dyads and were similar to those observed at the family of repeats of the wild-type oriP (Gahn and Schildkraut, Cell 58:527-535, 1989). In vitro experiments indicate that the viral trans-acting factor EBNA-1 contributes to efficient barrier formation at the tandem dyads as observed in the family of repeats of the wild-type oriP (V. Dhar and C. L. Schildkraut, Mol. Cell. Biol. 11:6268-6278, 1991). The tandem dyads thus appear to function in a manner similar to the family of repeats. There are significant structural differences between the family of repeats and tandem dyads. The relationship between the number and relative positions of EBNA-1 binding sites in relation to the functions of the family of repeats and the dyad symmetry element is discussed.     

Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids

Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.     

Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication.

The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequence requirements of the Epstein-Barr virus latent origin of DNA replication.

The Epstein-Barr virus (EBV) latent origin of DNA replication (oriP) is composed of two elements that contain binding sites for the sole viral gene product required for latent cycle replication, EBNA-1. One of these elements, region I, functions as an EBNA-1-dependent enhancer for RNA polymerase II-transcribed genes, may play a role in plasmid segregation, and is required for origin function in B cells latently infected with EBV. The second element, region II, contains or is very near the site of initiation of DNA replication. A genetic approach was taken to determine the contribution of the EBNA-1 binding sites in oriP to origin function. Although region I is required for the transient replication of plasmids bearing region II in EBV-infected B cells, a plasmid lacking region I but containing region II, was observed to replicate transiently in both D98/Raji and HeLa cells expressing EBNA-1. Thus, binding of EBNA-1 to region I is not absolutely required for the molecular events that lead to initiation of DNA replication at region II. Site-directed mutagenesis of the four EBNA-1-binding sites in region II, individually and in various combinations, demonstrated that only two EBNA-1-binding sites are required for region II function. The results obtained with these mutants, together with the analysis of the replicative ability of plasmids containing insertions between EBNA-1-binding sites, suggested that the spatial relationship of the two sites is critical. Mutants that contain only two EBNA-1-binding sites separated by 26 to 31 bp in region II were not maintained as plasmids over many cell generations and were greatly reduced in their ability to replicate transiently in D98/Raji cells. The EBNA-1-induced bending or untwisting of the DNA in EBNA-1-binding sites 1 and 4 in region II did not, however, demonstrate this spatial constraint. It may be concluded from these results that specific protein-protein interactions between EBNA-1 and/or between EBNA-1 and a cellular protein(s) are required for origin function.     

Coupling of mitotic chromosome tethering and replication competence in epstein-barr virus-based plasmids

The Epstein-Barr virus (EBV) replicates once per cell cycle and segregates with high efficiency yet does not encode the enzymes needed for DNA replication or the proteins required to contact mitotic spindles. The virus-encoded EBNA-1 (EBV nuclear antigen 1) and latent replication origin (oriP) are required for both replication and segregation. We developed a sensitive and specific fluorescent labeling strategy to analyze the interactions of both EBNA-1 with viral episomes and viral episomes with host chromosomes. This enabled investigation of the hypothesis that replication and chromosome tethering are linked through the EBNA-1 protein. We show that deleting EBNA-1 or oriP disrupts mitotic chromosome tethering but removing the dyad symmetry element of oriP does not. Microscopic and biochemical approaches demonstrated that an EBNA-1 mutant lacking residues 16 to 372 bound to oriP plasmids but did not support their mitotic chromosome association and that the mutant lost the ability of wild-type EBNA-1 to associate with interphase chromatin. Importantly, the transient-replication abilities of various mutant forms of EBV plasmids, including the mutant form with the EBNA-1 internal deletion, correlated directly with their chromosome-tethering abilities. These data lead us to propose that EBNA-1 recruits oriP-containing plasmids into chromatin subdomains in interphase nuclei to both engage the host replication machinery and enable the plasmids to adhere to host chromosomes to increase their segregation efficiency.     

The replicator of the Epstein-Barr virus latent cycle origin of DNA replication, oriP, is composed of multiple functional elements

Replication of the Epstein-Barr virus genome initiates at one of several sites in latently infected, dividing cells. One of these replication origins is close to the viral DNA maintenance element, and, together, this replication origin and the maintenance element are referred to as oriP. The replicator of oriP contains four binding sites for Epstein-Barr virus nuclear antigen 1 (EBNA-1), the sole viral protein required for the replication and maintenance of oriP plasmids. We showed previously that these EBNA-1 sites function in pairs and that mutational inactivation of one pair does not eliminate replicator function. In this study we characterized the contribution of each EBNA-1 site within the replicator and flanking sequences through the use of an internally controlled replication assay. We present evidence that shows that all four EBNA-1 sites are required for an oriP plasmid to be replicated in every cell cycle. Results from these experiments also show that the paired EBNA-1 binding sites are not functionally equivalent and that the low affinity of sites 2 and 3 compared to that of sites 1 and 4 is not essential for replicator function. Our results suggest that a host cell protein(s) binds sequences flanking the EBNA-1 sites and that interactions between EBNA-1 and this protein(s) are critical for replicator function. Finally, we present evidence that shows that the minimal replicator of oriP consists of EBNA-1 sites 3 and 4 and two copies of a 14-bp repeat that is present in inverse orientation flanking these EBNA-1 sites. EBNA-1 sites 1 and 2, together with an element(s) within nucleotides 9138 to 9516, are ancillary elements required for full replicator activity.