Establishment of an oriP replicon is dependent upon an infrequent, epigenetic event

Plasmids containing oriP, the latent origin of replication for Epstein-Barr virus, support efficient replication in selected cell clones when the vira l protein EBNA-1 is provided, being lost at a rate of 2 to 4% per cell gene ration after removal of selection (A. L. Kirchmaier and B. Sugden, J. Virol . 69:1280-1283, 1995; B. Sugden and N. Warren, Mel. Biol. Med. 5:85-94, 198 8). We refer to these plasmids as established replicons in that they suppor t efficient DNA synthesis and partitioning each cell cycle. Unexpectedly, w e have found that upon introduction of oriP plasmids into a population of E BNA-1-positive cells, oriP plasmids replicate but are lost precipitously fr om cells during 2 weeks posttransfection (>25% rate of loss per cell genera tion). Upon investigation of these disparate observations, we have found th at only 1 to 10% of cells transfected with an oriP plasmid expressing EBNA- 1 and hygromycin phosphotransferase give rise to drug-resistant clones in w hich the oriP replicon is established. A hereditable alteration in these dr ug-resistant cell clones, manifested at the genetic or epigenetic level, do es not underlie the establishment of oriP, as newly introduced oriP plasmid s replicate but are also lost rapidly from these cells. In addition, agenet ic alteration in the oriP plasmid is not responsible for establishment, as oriP plasmids isolated from an established cell clone, propagated in Escher ichia coil, and reintroduced into EBNA-1-positive cells are likewise establ ished inefficiently. Our findings demonstrate that oriP replicons are not i ntrinsically stable in EBNA-1-positive cell lines. Rather, the establishmen t of an oriP replicon is conferred upon the replicon by a stochastic, epige netic event that occurs infrequently and, therefore, is detected in only a minority of cells.