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

Abstract

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 DNAbinding 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.

The AT-hook DNA binding ability of the Epstein Barr virus EBNA1 protein is necessary for the maintenance of viral genomes in latently infected cells

“…We generated plasmids encoding these fusion proteins (p4880-H1 and p4881-LANA1(1–22) respectively), the OriP portion of the EBV genome and a gene encoding resistance to hygromycin (Figure 1C). Both the H1/EBNA1-DBD and the LANA1:1–22/EBNA1-DBD fusion proteins lack any AT-hook DNA-binding domain, but we confirmed previous results showing that both are able to maintain EBV-derived plasmids in 293 cells in the presence of selection, as are 293 cells harboring the parental p294-wtEBNA1 (Figure 1D) [20], [29]. …”

“…Interestingly, we and others have shown that the entire N-terminal half of EBNA1 can be replaced by proteins with or without AT-hook DNA-binding domains that maintain EBV-derived plasmids [20],[28],[29]. These seemingly contradictory findings together indicate an EBNA1-fusion protein must be able to bind mitotic chromosomes by some means in order to maintain EBV-derived plasmids in cells.…”

“…In addition, the entire N-terminal half of EBNA1 can be replaced by cellular proteins that contain AT-hook DNA-binding domains, such as HMGA1, and the fusion protein is able to mediate persistence of EBV-derived plasmids in cells [28]. However, other cellular and viral proteins that bind chromosomes, but lack AT-hook DNA-binding domains, such as the cellular protein Histone H1 or the first 22 amino acids of the LANA1 protein from Kaposi’s Sarcoma-associated Herpes Virus (KSHV) are also able to complement the loss of EBNA1’s LR1 and LR2 to maintain oriP replicons [20],[29]. …”

Recent technological updates and clinical applications of induced pluripotent stem cells

“…The EBNA-1/oriP association permits the persistence of plasmids in actively dividing human cells as multicopy episomes that attach to chromosomes during mitosis [34,35]. After multiple cell divisions, oriP/EBNA-based vectors are progressively lost from the targeted host cells.…”

Epstein–Barr virus latent antigens EBNA3C and EBNA1 modulate epithelial to mesenchymal transition of cancer cells associated with tumor metastasis

“…EBNA1 is a nuclear phosphoprotein which binds to the latent viral DNA replication origin and is responsible for maintenance of the viral genome in the EBV-positive cells after cell division [32–34]. EBNA1, LMP1, and LMP2A have earlier been shown to promote EMT [17, 35, 36].…”