Functional analyses of the EBNAl origin DNA binding protein of Epstein-Barr virus

The EBNA1 protein of Epstein-Barr virus (EBV) governs the replication and s egregation of the viral episomes in latently infected cells and transactiva tes the expression of other EBV latency proteins through direct interaction s with DNA sequences in the EBV latent origin of replication, oriP. To bett er understand how EBNA1 controls these processes, we have assessed the cont ribution of various EBNA1 sequences to its replication, segregation, and tr ansactivation functions. Here we show that EBNA1 residues 325 to 376 are re sponsible for the transactivation activity of EBNA1. This region coincides with the DNA looping domain previously shown to mediate interactions at a d istance between DNA-bound EBNA1 molecules. The same residues mediate DNA se gregation but have no apparent role in DNA replication, indicating that the replication and transcription activation activities of EBNA1 are distinct. The acidic C-terminal tail of EBNA1 was not found to contribute to replica tion, transactivation, or segregation. We have also investigated the functi onal significance of two structural motifs within the DNA binding and dimer ization domains of EBNA1, the proline loop and the WF motif. Although the a mino acids in these motifs do not directly contact the DNA, both of these m otifs were found to contribute to EBNA1 functions by increasing the DNA-bin ding ability of EBNA1. Mechanisms by which DNA binding is stimulated by the se motifs are discussed.