Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein-Barr virus

Epstein-Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP-dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co-immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.     

The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency

Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research.     

Telomeric proteins regulate episomal maintenance of Epstein-Barr virus origin of plasmid replication

Episomal maintenance and DNA replication of EBV origin of plasmid replication (OriP) plasmid maintenance is mediated by the viral encoded origin binding protein, EBNA1, and unknown cellular factors. We found that telomeric repeat binding factor 2 (TRF2), TRF2-interacting protein hRap1, and the telomere-associated poly(ADP-ribose) polymerase (Tankyrase) bound to the dyad symmetry (DS) element of OriP in an EBNA1-dependent manner. TRF2 bound cooperatively with EBNA1 to the three nonamer sites (TTAGGGTTA), which resemble telomeric repeats. Mutagenesis of the nonamers reduced plasmid maintenance function and increased plasmid sensitivity to genotoxic stress. DS affinity-purified proteins possessed poly(ADP-ribose) polymerase (PARP) activity, and EBNA1 was subject to NAD-dependent posttranslational modification in vitro. OriP plasmid maintenance was sensitive to changes in cellular PARP/Tankyrase activity. These findings imply that telomere-associated proteins regulate OriP plasmid maintenance by PAR-dependent modifications.     

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.     

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.     

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.     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.     

Two domains of the epstein-barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding

The EBNA1 (for Epstein-Barr nuclear antigen 1) protein of Epstein-Barr virus governs the replication and partitioning of the viral genomes during latent infection by binding to specific recognition sites in the viral origin of DNA replication. The crystal structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homologous to the DNA binding domain of the papillomavirus E2 protein, and a flanking domain, which mediated all the observed sequence-specific contacts. To test the possibility that the EBNA1 core domain plays a role in sequence-specific DNA binding not revealed in the crystal structure, we examined the effects of point mutations in potential hydrogen bond donors located in an alpha-helix of the EBNA1 core domain whose structural homologue in E2 mediates sequence-specific DNA binding. We show that these mutations severely reduce the affinity of EBNA1 for its recognition site, and that the core domain, when expressed in the absence of the flanking domain, has sequence-specific DNA binding activity. Flanking domain residues were also found to contribute to the DNA binding activity of EBNA1. Thus, both the core and flanking domains of EBNA1 play direct roles in DNA recognition.     

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.     

Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

The specific binding of HeLa cell factors to DNA sequences at the Epstein-Barr virus (EBV) latent origin of DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular proteins protected at least five discrete regions of the DNA replication origin. The viral protein required for EBV plasmid replication, EBV nuclear antigen 1 (EBNA-1), binds to specific sequences within the origin region. The HeLa cell proteins competed with EBNA-1 for binding to EBV origin DNA in vitro, leading to the possibility that these cellular proteins regulate EBV DNA replication by displacing EBNA-1 at the origin sites.     

Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.