Maintenance of Epstein-Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA el ement, oriP, enables plasmids to persist in dividing human cells as multico py episomes that attach to chromosomes during mitosis, In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the b asic domains of EBNA-1 within amino acids 1-89 and 323-386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379-641 ), which includes the nuclear localization signal and DNA-binding domain, d oes not associate with mitotic chromosomes or retain oriP plasmid DNA in di viding cell nuclei, but does enable the accumulation of replicated oriP-con taining plasmid DNA in transient replication assays. The importance of chro mosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1-378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-1 amino acids 1-90 or histone H1-2 cou ld substitute for EBNA-1 amino acids 1-378 in mediating more efficient accu mulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly s upport the hypothesis that mitotic chromosome association is a critical fac tor for episome maintenance, The replacement of 60% of EBNA-1 with cell pro tein is a significant step toward eliminating the need for noncellular prot ein sequences in the maintenance of episomal DNA in human cells.