Abstract
OriP, the latent origin of Epstein-Barr virus (EBV), consists of two essential elements: the dyad symmetry (DS) and the family of repeats (FR). The function of these elements has been predominantly analyzed in plasmids transfected into transformed cells. Here, we examined the molecular functions of DS in its native genomic context and at an ectopic position in the mini-EBV episome. Mini-EBV plasmids contain 41% of the EBV genome including all information required for the proliferation of human B cells. Both FR and DS function independently of their genomic context. We show that DS is the most active origin of replication present in the mini-EBV genome regardless of its location, and it is characterized by the binding of the origin recognition complex (ORC) allowing subsequent replication initiation. Surprisingly, the integrity of oriP is not required for the formation of the pre-replicative complex (pre-RC) at or near DS. In addition we show that initiation events occurring at sites other than the DS are also limited to once per cell cycle and that they are ORC-dependent. The deletion of DS increases initiation from alternative origins, which are normally used very infrequently in the mini-EBV genome. The sequence-independent distribution of ORC-binding, pre-RC-assembly, and initiation patterns indicates that a large number of silent origins are present in the mini-EBV genome. We conclude that, in mini-EBV genomes lacking the DS element, the absence of a strong ORC binding site results in an increase of ORC binding at dispersed sites.
Highlights
Epstein-Barr virus (EBV) infects human B-cells and establishes a persistent latent infection
In this report we studied the efficiency of pre-replicative complex (pre-RC) assembly and site-specific replication initiation in the context of mini-EBV genomes
Our data provide evidence that in mini-EBV genomes dyad symmetry (DS) is the predominant site of origin recognition complex (ORC)-binding and pre-RC assembly and the preferred initiation start site regardless of the cellular background (Figs. 2 to 6 and S1, S2)
Summary
EBV infects human B-cells and establishes a persistent latent infection. The viral genome is maintained autonomously in proliferating cells and each viral episome is replicated once per cell cycle during S phase [1,2]. FR consists of an array of twenty imperfect 30 bp repeats (each containing an EBNA1 binding site) and contributes to the stable maintenance of oriP-containing episomes by preventing their loss during cell division. DS contains four EBNA1 binding sites arranged in pairs and is the only part of oriP required for DNA replication initiation in small plasmids. During S phase pre-RCs are lost either by the activation of origins or by passing replication forks [20]. These findings led to the hypothesis that oriP is regulated to chromosomal origins, an assumption further supported by the observation that small oriP-plasmids do not replicate in an Orc2-mutant cell line and their replication is inhibited by the overexpression of geminin [21]
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