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

摘要

EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1--89 and 323--386 as critical for chro- mosome binding. In contrast, the EBNA-1 C terminus (amino acids 379--641), which includes the nuclear localization signal and DNA- binding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in tran- sient replication assays. The importance of chromosome associa- tion 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-Ⅰ amino acids 1--90 or histone H1--2 could substitute for EBNA-1 amino acids 1--378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60/100 of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells.