INITIATION OF LATENT DNA-REPLICATION IN THE EPSTEIN-BARR-VIRUS GENOMECAN OCCUR AT SITES OTHER THAN THE GENETICALLY DEFINED ORIGIN

Our laboratory has previously shown that replication of a small plasmi d, p174, containing the genetically defined Epstein-Barr virus (EBV) l atent 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 similar to 170-kb EBV genomes i n four latently infected cell lines that uses two dimensional gel repl icon mapping. Initiation was detected at oriP in all EBV genomes exami ned; however, some replication forks appear to originate from alternat ive initiation sites. In addition, pausing of replication forks was ob served at the two clusters of EBV nuclear antigen 1 binding sites with in oriP and at or near two highly expressed viral genes 0.5 to 1 kb up stream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV geno me, 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 di stributed over a broad region to the left of oriP. This delocalized pa ttern of initiation resembles initiation of replication in several wel l-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with whi ch to investigate factors influencing the selection of replication ini tiation and termination sites in mammalian cells.