Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication

The Epstein-Barr virus latent origin of replication (oriP) requires only one viral protein, the Epstein-Barr virus nuclear antigen 1 (EBNA-1), for activity. oriP consists of two spatially separated, essential sequence elements, regions I and II, both of which contain multiple EBNA-1-binding sites. Region II contains, or is close to, the site at which DNA synthesis initiates. The role of region I, a transcriptional enhancer in cells that express EBNA-1, in replication is not understood. To determine how the binding of EBNA-1 to sites in region II leads to the initiation of DNA synthesis and to investigate the role of region I, EBNA-1 has been overproduced in insect cells by using a baculovirus vector and purified to homogeneity, and the interaction of EBNA-1 with oriP has been examined. Footprinting experiments demonstrated that EBNA-1 binds to oriP in a sequence-specific manner and bends or untwists the DNA at two symmetry-related sites in region II. Distortion of region I by EBNA-1 was not detected, suggesting that differences in the spacing of binding sites in regions I and II and resulting protein-protein interactions underlie differences in their biological properties. KMnO4 footprinting experiments did not reveal significant single-stranded structures in region II, suggesting that cellular proteins may recognize the EBNA--region II complex and unwind the DNA duplex. Region I did not quantitatively or qualitatively alter the interaction of EBNA-1 with region II. The contribution of an A + T-rich sequence in region II to replication was investigated by a mutational analysis. The results indicated that the overall A + T-rich nature of this sequence is not essential for replication of oriP-bearing plasmids. Nuclease protection experiments performed with these mutagenized plasmids provided additional evidence for protein-protein interactions in region II.