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

Abstract

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.

Figure 1

The EBV genome and the latent origin of plasmid replication (oriP). (A) The genome of EBV is depicted here in its covalently closed circular form of ~165kbp. The original genomic fragments produced by BamHI digestion are labeled with letters on the interior. Promoters, transcription units, and gene products of the latent cycle are labeled on the exterior as arrows, dashed lines, and open boxes, respectively. In bold boxes are the lytic origin of DNA synthesis (oriLyt), the latent origin of DNA synthesis (oriP), the terminal repeats (TR), and the ~14kbp region shown to support initiation of DNA synthesis in the Raji strain of EBV (Raji ori). (B) The origin of plasmid replication (oriP) is composed of the Family of Repeats (FR) and the Dyad Symmetry (DS) element. The Rep* element is located 240bp downstream of DS, and the original mapping of this element is shown as a lightly shaded box. EBNA1-binding sites are depicted as open boxes in all three cis elements; the dyad present in DS is denoted by the head-to-head arrows above it. The base positions of each of these elements are listed below them with respect to the sequence of the B95–8 strain of EBV. (C) A more detailed depiction of the DS element. The two pairs of EBNA1-binding sites of DS have an intra-pair spacing of 21bp from the center of one site to the center of the paired site, and an inter-pair spacing of 33bp between the centers of the adjacent binding sites. Nine base pair binding sites for the TTAGGG-repeat Binding Factor 2 (TRF2), also referred to as 'nonamers,' flank the pairs of EBNA1-binding sites and are denoted by the shaded boxes. The arrows above the TRF2-binding sites denote the N-to-C terminal orientation that TRF2 would use to bind to these sites.

Figure 2

A Domain-Based Model and Partial Structural Representation of Epstein-Barr Nuclear Antigen 1 (EBNA1). EBNA1 is depicted as modules corresponding to functional domains identified by mutational or deletional analyses. Amino acid (AA) positions are denoted below the junction of adjacent domains. G-R denotes a glycine-arginine rich region within the linking regions 1 (AA40–89) and 2 (AA325–379). Two regions composed of more unique amino acids within LR1 and LR2 are denoted as Unique Region 1 (UR-1) and Unique Region 2 (UR-2), respectively. G-G-A denotes a glycine-glycine-alanine region that composes over a third of the molecule. NLS denotes a nuclear localization signal. The DNA-binding and dimerization domain is labeled according to its functions, and has been crystallized and its structure determined by x-ray diffraction. The acidic tail is rich in aspartic and glutamic acids. Functions attributed to EBNA1 are denoted in rectangles below the modular depiction of EBNA1; additional proteins shown to interact with specific regions of EBNA1 are denoted in rounded rectangles above it.

Figure 3

A sequential model of the licensing process for DNA synthesis at an EBNA1-dependent origin. (A) A pair of EBNA1 dimers binds to a pair of 16bp binding sites with a precise 21bp center-to-center spacing, placing the dimers on the same helical face of the DNA. The binding event bends the DNA toward the DNA-binding and dimerization domain in an additive manner and in the same direction. The TTAGGG-repeat Binding Factor 2 (TRF2) is found as a dimer in the cell, and one monomer can bind to a 9bp element that flanks the pairs of EBNA1-binding sites in wtDS. The binding of TRF2 to these elements (also termed nonamers) permits the cooperative binding of EBNA1 to its binding sites, thereby increasing EBNA1’s apparent affinity. In addition, ORC2–6 were found to be bound to such an EBNA1-dependent origin throughout the cell cycle by Chromatin Immunoprecipitation (ChIP). (B) ORC1 has been shown to interact with EBNA1-dependent origins in the presence and absence of the TRF2 binding sites, although ORC1’s association with these origins is likely enhanced by the interaction of it’s BAH domain with the N-terminal basic domain of TRF2. Cdc6 is also likely recruited to the origin in G1/S. (C) Taken together, these assembled proteins at the EBNA1-dependent origin allow the subsequent recruitment of, and likely the reiterative loading of, the putative replicative helicase MCM2–7 as a head-to-head double hexamer, possibly in a complex with the regulatory protein Cdt1.