Maintenance of Epstein-Barr virus-derived episomal vectors in the murine Sp2/0 myeloma cell line is dependent upon exogenous expression of human EBP2.

Vectors carrying the origin of replication (oriP) and driving expression of the EBNA-1 protein from Epstein-Barr virus (EBV) replicate as extrachromosomal episomes in human cells. Whether these vectors can be maintained as episomes in murine cells is still controversial. Here we demonstrate that EBNA-1 expression alone was unable to maintain episomal expression of an EBV-based vector in the murine Sp2/0 cell line. However, we were able to obtain long-term episome maintenance in Sp2/0 cells after exogenously expressing human EBP2 by genetic engineering. Our results provide further evidence for the fundamental role of human EBP2 in episomal maintenance of EBV-based vectors. Moreover, we demonstrate that EBV-based vectors can be successfully used in cells presumably incompetent for episomal maintenance.     

Construction of Epstein-Barr virus-based expression vector containing mini-oriP.

Epstein-Barr virus (EBV)-based vectors are extrachromosomal vectors carrying a replicational origin, oriP (about 2200 bp) and a replication initiation factor (EBNA-1) which are sufficient for autonomous replication. Because one disadvantage of these vectors is their large sizes, we examined the effect of partial deletion of oriP on the effectiveness of the EBV-based vectors, using an enhanced green fluorescent protein (EGFP) as a reporter to monitor gene expression. Results indicated that 954 bp-deleted mini-oriP is useful in primate cells since the vector showed high efficiency of stable transfection, a high ratio of EGFP-positive cells, and high recovery of intact plasmid DNA from transfected cells.     

Sequences adjacent to oriP improve the persistence of Epstein-Barr virus-based episomes in B cells

Epstein-Barr virus (EBV) oriP and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spans oriP and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing oriP alone.     

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.     

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.     

Induction of efficient differentiation and survival of porcine neonatal pancreatic cell clusters using an EBV-based plasmid expressing HGF

Porcine neonatal pancreatic cell clusters (NPCCs) have been actively studied as a source of pancreatic stem cell transplantation for the treatment of diabetes. In this study, the hepatocyte growth factor (HGF) gene was cloned in an Epstein-Barr virus (EBV)-based plasmid vector (pEBVHGF) and the effects of the HGF expression on the survival and differentiation of NPCCs were analysed. For comparison, pHGF was constructed by deleting EBNA-1 and OriP from pEBVHGF. The expression of HGF, as measured by ELISA, lasted longer when pEBVHGF was used than when pHGF was used. C-Met phosphorylation co-related with the expression of HGF in the transfected NPCCs. Immunocytochemistry experiments showed that NPCCs showed a higher and longer expression of insulin when they were transfected with pEBVHGF than with pHGF. Moreover, a greater number of NPCCs survived for a longer period after they were transfected with pEBVHGF than when they were transfected with pHGF. Taken together, these results indicate that transfecting NPCCs with the HGF gene using an EBV-based plasmid is a more effective method of inducing differentiation to beta cells and enhancing survival than using a conventional plasmid. Therefore, it may be possible to use EBV-based plasmids to modify pancreatic stem cells for xenotransplantation.     

Non-replicating Epstein-Barr virus-based plasmids extend gene expression and can improve gene therapy in vivo

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.     

Interaction of the lymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with its DNA-binding sites.

The Epstein-Barr virus (EBV) nuclear antigen EBNA-1 plays an integral role in the maintenance of latency in EBV-infected B lymphocytes. EBNA-1 binds to sequences within the plasmid origin of replication (oriP). It is essential for the replication of the latent episomal form of EBV DNA and may also regulate the expression of the EBNA group of latency gene products. We have used sequence-specific DNA-binding assays to purify EBNA-1 away from nonspecific DNA-binding proteins in a B-lymphocyte cell extract. The availability of this eucaryotic protein has allowed an examination of the interaction of EBNA-1 with its specific DNA-binding sites and an evaluation of possible roles for the different binding loci within the EBV genome. DNA filter binding assays and DNase I footprinting experiments showed that the intact Raji EBNA-1 protein recognized the two binding site loci in oriP and the BamHI-Q locus and no other sites in the EBV genome. Competition filter binding experiments with monomer and multimer region I consensus binding sites indicated that cooperative interactions between binding sites have relatively little impact on EBNA-1 binding to region I. An analysis of the binding parameters of the Raji EBNA-1 to the three naturally occurring binding loci revealed that the affinity of EBNA-1 for the three loci differed. The affinity for the sites in region I of oriP was greater than the affinity for the dyad symmetry sites (region II) of oriP, while the physically distant region III locus showed the lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can lowest affinity. This arrangement may provide a mechanism whereby EBNA-1 can mediate differing regulatory functions through differential binding to its recognition sequence.     

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.     

Efficient method for expressing transgenes in nonhuman primate embryos using a stable episomal vector

Transgenesis in the nonhuman primate can enhance the study of human biology by providing animal models for the study of primate-specific physiology, pathophysiology, and embryonic development. Progress with this technology has been hindered by the inherent inefficiency of transgenesis, transgene silencing, and practical restrictions on the production of sufficient pronuclear stage nonhuman primate zygotes. We have developed a novel technique using an Epstein Barr virus (EBV)-based episomal vector to produce rhesus monkey (Macaca mulatta) embryos expressing a transgene. Plasmid DNA containing the latent origin of replication, oriP, and Epstein Barr Nuclear Antigen-1 (EBNA-1) of EBV, as well as a CMV IE-enhanced green fluorescent protein (eGFP) expression cassette, was introduced into rhesus embryos by direct pronuclear microinjection. We detected eGFP in early cleavage stage embryos (4-8 cell) and throughout the duration of culture (day 8-9 blastocysts) by epifluorescent microscopy. A 50% transduction rate was obtained with the EBV-based vector. Microinjected embryos expressed eGFP and retained their developmental capacity as evidenced by development to the blastocyst stage. EBV-based vectors present a novel and efficient means of delivering transgenes for the study of the molecular control of primate embryonic development.     

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.     

Establishment of an oriP replicon is dependent upon an infrequent, epigenetic event

Plasmids containing oriP, the latent origin of replication for Epstein-Barr virus, support efficient replication in selected cell clones when the viral protein EBNA-1 is provided, being lost at a rate of 2 to 4% per cell generation after removal of selection (A. L. Kirchmaier and B. Sugden, J. Virol. 69:1280-1283, 1995; B. Sugden and N. Warren, Mol. Biol. Med. 5:85-94, 1988). 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). Upon investigation of these disparate observations, we have found that only 1 to 10% of cells transfected with an oriP plasmid expressing EBNA-1 and hygromycin phosphotransferase give rise to drug-resistant clones in which the oriP replicon is established. A hereditable alteration in these drug-resistant cell clones, manifested at the genetic or epigenetic level, does not underlie the establishment of oriP, as newly introduced oriP plasmids replicate but are also lost rapidly from these cells. In addition, a genetic alteration in the oriP plasmid is not responsible for establishment, as oriP plasmids isolated from an established cell clone, propagated in Escherichia coli, and reintroduced into EBNA-1-positive cells are likewise established inefficiently. Our findings demonstrate that oriP replicons are not intrinsically stable in EBNA-1-positive cell lines. Rather, the establishment of an oriP replicon is conferred upon the replicon by a stochastic, epigenetic event that occurs infrequently and, therefore, is detected in only a minority of cells.     

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.     

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.     

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.