Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates latent-phase DNA replication by an unknown mechanism that involves binding to four recognition sites in the dyad symmetry (DS) element of the viral latent origin of DNA replication. Since EBV episomes are assembled into nucleosomes, we have examined the ability of Epstein-Barr virus nuclear antigen 1 (EBNA1) to interact with the DS element when it is assembled into a nucleosome core particle. EBNA1 bound to its recognition sites within this nucleosome, forming a ternary complex, and displaced the histone octamer upon competitor DNA challenge. The DNA binding and dimerization region of EBNA1 was sufficient for nucleosome binding and destabilization. Although EBNA1 was able to bind to nucleosomes containing two recognition sites from the DS element positioned at the edge of the nucleosome, nucleosome destabilization was only observed when all four sites of the DS element were present. Our results indicate that the presence of a nucleosome at the viral origin will not prevent EBNA1 binding to its recognition sites. In addition, since four EBNA1 recognition sites are required for both nucleosome destabilization and efficient origin activation, our findings also suggest that nucleosome destabilization by EBNA1 is important for origin activation.

Mechanistic studies on the DNA linking activity of Epstein-Barr nuclear antigen 1.

The DNA replication, plasmid segregation and transactivation functions of Epstein-Barr nuclear antigen 1 (EBNA1) require the binding of EBNA1 to specific DNA recognition sites in the two non-contiguous functional elements of the Epstein-Barr virus latent origin of replication, oriP . EBNA1 molecules bound to these elements interact with each other resulting in the formation of looped individual DNA molecules and multiply linked DNA molecules. We have developed a glycerol gradient sedimentation assay suitable for quantitative analysis of the DNA linking activity of EBNA1 and used it to investigate the contribution of EBNA1 residues to the linking interaction and the mechanism of the interaction. Using overlapping internal deletion mutants, we found that two regions of EBNA1 can cause DNA linking, amino acids 40-100 and 327-377, but that the stabilities of the linked complexes formed by the two regions differ dramatically; only complexes formed through the latter region are stable to glycerol gradient sedimentation analysis. Mechanistic studies using EBNA1 in combination with GAL4-EBNA1 fusion proteins showed that linking interactions mediated by residues 327-377 are homotypic. Our results also suggest that only the DNA-bound form of EBNA1 participates in the protein-protein interactions seen in DNA linking.

EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance.

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient replication of oriP plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.