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 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 chromosome 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 transient replication assays. The importance of chromosome 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-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% 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.
Hung
S C
SC
Program in Virology, Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Kang
M S
MS
Kieff
E
E
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D025741:HMGA1a Protein; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007399:Interphase; D008938:Mitosis; D010957:Plasmids / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D019309:Epstein-Barr Virus Nuclear Antigens; D006609:High Mobility Group Proteins; D006657:Histones; D014157:Transcription Factors; D025741:HMGA1a Protein; C417029:EBV-encoded nuclear antigen 1