EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient replication of oriP plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.     

Cooperative assembly of EBNA1 on the Epstein-Barr virus latent origin of replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates DNA replication by binding to multiple copies of its 18-bp recognition sequence present in the Epstein-Barr virus latent origin of DNA replication, oriP. Using electrophoretic mobility shift assays, we have localized the minimal DNA binding domain of EBNA1 to between amino acids 470 and 607. We have also demonstrated that EBNA1 assembles cooperatively on the dyad symmetry subelement of oriP and that this cooperative interaction is mediated by residues within the minimal DNA binding and dimerization domain of EBNA1.     

The amino terminus of Epstein-Barr Virus (EBV) nuclear antigen 1 contains AT hooks that facilitate the replication and partitioning of latent EBV genomes by tethering them to cellular chromosomes

During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.     

The DNA segregation mechanism of Epstein-Barr virus nuclear antigen 1

Latent Epstein–Barr virus (EBV) genomes are maintained in human cells as low copy number episomes that are thought to be partitioned by attachment to the cellular mitotic chromosomes through the viral EBNA1 protein. We have identified a human protein, EBP2, which interacts with the EBNA1 sequences that govern EBV partitioning. Here we show that, in mitosis, EBP2 localizes to the condensed cellular chromosomes producing a staining pattern that is indistinguishable from that of EBNA1. The localization of EBNA1 proteins with mutations in the EBP2 binding region was also examined. An EBNA1 mutant (Δ325–376) disrupted for EBP2 binding and segregation function was nuclear but failed to attach to the cellular chromosomes in mitosis. Our results indicate that amino acids 325–376 mediate the binding of EBNA1 to mitotic chromosomes and strongly suggest that EBNA1 mediates EBV segregation by attaching to EBP2 on the cellular mitotic chromosomes.     

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

The genome of Epstein-Barr Virus (EBV) and plasmid derivatives of it are among the most efficient extrachromosomal replicons in mammalian cells. The latent origin of plasmid replication (oriP), when supplied with the viral Epstein-Barr Nuclear Antigen 1 (EBNA1) in trans, provides efficient duplication, partitioning and maintenance of plasmids bearing it. In this review, we detail what is known about the viral cis and trans elements required for plasmid replication. In addition, we describe how the cellular factors that EBV usurps are used to complement the functions of the viral constituents. Finally, we propose a model for the sequential assembly of an EBNA1-dependent origin of DNA synthesis into a pre-Replicative Complex (pre-RC), which functions by making use only of cellular enzymatic activities to carry out the replication of the viral plasmid.     

EBNA1 distorts oriP, the Epstein-Barr virus latent replication origin.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed.     

Identification of EBNA1 amino acid sequences required for the interaction of the functional elements of the Epstein-Barr virus latent origin of DNA replication.

Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin, oriP. This activation involves the direct interaction of EBNA1 dimers with multiple sites within the two noncontiguous functional elements of the origin, the family of repeats (FR) element and the dyad symmetry (DS) element. The efficient interaction of EBNA1 dimers bound to these two elements in oriP results in the formation of DNA loops in which the FR and DS elements are bound together through EBNA1. In order to elucidate the mechanism by which EBNA1 induces oriP DNA looping, we have investigated the DNA sequences and EBNA1 amino acids required for EBNA1-mediated DNA looping. Using a series of truncation mutants of EBNA1 produced in baculovirus and purified to apparent homogeneity, we have demonstrated that the EBNA1 DNA binding and dimerization domain is not sufficient to mediate oriP DNA looping and that an additional region(s) located between amino acids 346 and 450 is required. Single EBNA1-binding sites, separated by 930 bp of plasmid DNA, were also shown to support EBNA1-mediated looping, indicating that the formation of large EBNA1 complexes, such as those observed on oriP FR and DS elements, is not a requirement for looping.     

Mechanistic studies on the DNA linking activity of Epstein-Barr nuclear antigen 1.

The DNA replication, plasmid segregation and transactivation functions of Epstein-Barr nuclear antigen 1 (EBNA1) require the binding of EBNA1 to specific DNA recognition sites in the two non-contiguous functional elements of the Epstein-Barr virus latent origin of replication, oriP . EBNA1 molecules bound to these elements interact with each other resulting in the formation of looped individual DNA molecules and multiply linked DNA molecules. We have developed a glycerol gradient sedimentation assay suitable for quantitative analysis of the DNA linking activity of EBNA1 and used it to investigate the contribution of EBNA1 residues to the linking interaction and the mechanism of the interaction. Using overlapping internal deletion mutants, we found that two regions of EBNA1 can cause DNA linking, amino acids 40-100 and 327-377, but that the stabilities of the linked complexes formed by the two regions differ dramatically; only complexes formed through the latter region are stable to glycerol gradient sedimentation analysis. Mechanistic studies using EBNA1 in combination with GAL4-EBNA1 fusion proteins showed that linking interactions mediated by residues 327-377 are homotypic. Our results also suggest that only the DNA-bound form of EBNA1 participates in the protein-protein interactions seen in DNA linking.     

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.     

Replication from oriP of Epstein-Barr virus requires exact spacing of two bound dimers of EBNA1 which bend DNA

oriP is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports replication and stable maintenance of plasmids in human cells that contain EBV-encoded protein EBNA1. Plasmids that depend on oriP are replicated once per cell cycle by cellular factors. The replicator of oriP is an approximately 120-bp region called DS which depends on either of two pairs of closely spaced EBNA1 binding sites. Here we report that changing the distance between the EBNA1 sites of a functional pair by inserting or deleting 1 or 2 bp abolished replication activity. The results indicated that, while the distance separating the binding sites is critical, the specific nucleotide sequence between them is unlikely to be important. The use of electrophoretic mobility shift assays to investigate binding by EBNA1 to the sites with normal or altered spacing revealed that EBNA1 induces DNA to bend significantly when it binds, with the center of bending coinciding with the center of binding. EBNA1 binding to a functional pair of sites which are spaced 21 bp apart center to center and which thus are in helical phase induces a larger symmetrical bend, which based on electrophoretic mobility approximates the sum of two separate EBNA1-induced DNA bends. The results imply that replication from oriP requires a precise structure in which DNA forms a large bend around two EBNA1 dimers.     

Establishment of latent Epstein-Barr virus infection and stable episomal maintenance in murine B-cell lines

Epstein-Barr virus (EBV) is a strict human pathogen for which no small animal models exist. Plasmids that contain the EBV plasmid origin of replication, oriP, and express EBV nuclear antigen 1 (EBNA1) are stably maintained extrachromosomally in human cells, whereas these plasmids replicate poorly in rodent cells. However, the ability of oriP and EBNA1 to maintain the entire EBV episome in proliferating rodent cells has not been determined. Expression of the two human B-cell receptors for EBV on the surfaces of murine B cells allows efficient viral entry that leads to the establishment of latent EBV infection and long-term persistence of the viral genome. Latent gene expression in these cells resembles the latency II profile in that EBNA1 and LMP1 can be detected whereas EBNA2 and the EBNA3s are not expressed.