In latent Epstein-Barr virus infection, the viral EBNA1 protein binds to specific sites in the viral origin of DNA replication, oriP, to activate the initiation of DNA replication, enhance the expression of other viral latency proteins, and partition the viral episomes during cell division. The DNA binding domain of EBNA1 is required for all three function, and a Gly-Arg-rich sequence between amino acids 325 and 376 is required for both the transcriptional activation and partitioning functions. We have used mutational analysis to identify additional EBNA1 sequences that contribute to EBNA1 functions. We show that EBNA1 amino acids 8 to 67 contribute to, but are not absolutely required for, EBNA1 replication, partitioning, and transcriptional activation functions. A Gly-Arg-rich sequence (amino acids 33 to 53) that is similar to that of amino acids 325 to 376 and lies within the 8-to-67 region was not responsible for the functional contributions of residues 8 to 67, since deletion of amino acids 34 to 52 alone did not affect EBNA1 functions. We also found that deletion of amino acids 61 to 83 eliminated the transcriptional activity of EBNA1 without affecting partitioning. This mutant also exhibited an increased replication efficiency that resulted in the maintenance of oriP plasmids at a copy number approximately fourfold higher than for wild-type EBNA1. The results indicate that the three EBNA1 functions have overlapping but different sequence requirements. Transcriptional activation requires residues 61 to 83 and 325 to 376 and is stimulated by residues 8 to 67; partitioning requires residues 325 to 376 and is stimulated by residues 8 to 67; and replication involves redundant contributions of both the 325-to-376 and 8-to-67 regions.Epstein-Barr virus (EBV) persists in the nuclei of latently infected human B lymphocytes as double-stranded circular DNA episomes (51). These episomes undergo one round of DNA replication per cell cycle and are efficiently partitioned during cell division, enabling the viral genomes to be maintained at a constant copy number (1,32,53,54). The replication and partitioning of the EBV episomes require two viral components: the latent origin of DNA replication, oriP, and the EBNA1 protein (54). EBV DNA replication initiates within the dyad symmetry (DS) element of oriP, which contains four EBNA1 binding sites (17,37,40,50). The partitioning of the episomes involves EBNA1 binding to the family-of-repeats (FR) element of oriP, which contains 20 EBNA1 recognition sites (32,40). In addition, EBNA1 binding to the FR element enhances the expression of other viral latency proteins (16, 39).The mechanism by which EBNA1 binds to its recognition sites in oriP is reasonably well understood. EBNA1 binds to each site as a dimer, and the dimers assemble cooperatively on the four sites in the DS element (15, 21, 47). EBNA1 amino acids 459 to 607 are responsible for DNA binding and dimerization, and the structural basis for DNA recognition and dimerization has been revealed through X-ray crystallography...
The EBNA1 protein of Epstein±Barr virus (EBV) mediates the partitioning of EBV episomes and EBVbased plasmids during cell division by a mechanism that appears to involve binding to the cellular EBP2 protein on human chromosomes. We have investigated the ability of EBNA1 and the EBV segregation element (FR) to mediate plasmid partitioning in Saccharomyces cerevisiae. EBNA1 expression alone did not enable the stable segregation of FR-containing plasmids in yeast, but segregation was rescued by human EBP2. The reconstituted segregation system required EBNA1, human EBP2 and the FR element, and functionally replaced a CEN element. An EBP2 binding mutant of EBNA1 and an EBNA1 binding mutant of EBP2 each failed to support FR-plasmid partitioning, indicating that an EBNA1±EBP2 interaction is required. The results provide direct evidence of the role of hEBP2 in EBNA1-mediated segregation and demonstrate that heterologous segregation systems can be reconstituted in yeast.
Epstein-Barr virus (EBV) genomes persist indefinitely in latently infected human cells, in part due to their ability to stably segregate during cell division. This process is mediated by the viral EBNA1 protein, which tethers the viral episomes to the cellular mitotic chromosomes. We have previously identified a mitotic chromosomal protein, human EBNA1 binding protein 2 (hEBP2), which binds to EBNA1 and enables EBNA1 to partition EBV-based plasmids in Saccharomyces cerevisiae. Using an RNA silencing approach, we show that hEBP2 is essential for the proliferation of human cells and that repression of hEBP2 severely decreases the ability of EBNA1 and EBV-based plasmids to bind mitotic chromosomes. When expressed in yeast, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin as in human cells, and using yeast temperature-sensitive mutant strains, we found that the attachment of hEBP2 to mitotic chromosomes was dependent on the Ipl1 kinase. Both RNA silencing of the Ipl1 orthologue in human cells (Aurora B) and specific inhibition of the Aurora B kinase activity with a small molecule confirmed a role for this kinase in enabling hEBP2 binding to human mitotic chromosomes, suggesting that this kinase can regulate EBV segregation.Epstein-Barr virus (EBV) infects most people worldwide and stably persists for the life of the host through latent infection of B lymphocytes and epithelial cells (40). Due to the immortalization effects of the viral latency proteins, latent infection predisposes the host to develop a variety of malignancies. Only one viral protein, EBNA1, is required to maintain the viral episomes in proliferating latently infected cells, as it plays important roles in both the replication and the segregation of the EBV episomes during cell division (51). EBNA1 contributes to DNA replication by binding to specific sequences in the dyad symmetry element of the latent origin of replication, oriP, while the segregation of the EBV episomes requires EBNA1 binding to the 20 tandem recognition sites in the family of repeats (FR) element of oriP (30, 39). The interaction of EBNA1 with the dyad symmetry and FR sequences occurs through the DNA binding and dimerization domain of EBNA1, which is located between amino acids 459 and 607 (2, 45). In addition to this domain, the replication and segregation functions of EBNA1 require N-terminal sequences of EBNA1; replication appears to involve multiple redundant regions of the EBNA1 N terminus, while segregation function is absolutely dependent on the Gly-Arg-rich sequence between amino acids 325 and 376 and moderately affected by amino acids 8 to 67 (33,42,49).The concept that EBNA1 mediated the segregation of EBV episomes by tethering them to the cellular mitotic chromosomes stemmed from observations that EBNA1, EBV episomes, and oriP-containing constructs were all tightly associated with mitotic chromosomes and that the association of oriP plasmids with mitotic chromosomes was EBNA1 dependent (17,20,27,38,43). Mutational analyses of EBNA1 a...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.