Epstein-Barr virus (EBV) is a herpesvirus which is associated with several malignancies. A central aspect of the life cycle and pathogenesis of EBV is a latent state of infection in which the viral chromosome is maintained as an episome. A single EBV-encoded protein, EBNA1, allows the EBV chromosome to be maintained by binding to oriP, where it directs DMAreplication to initiate and where it also acts to prevent the EBV chromosome from being lost from mitotically active cells. Studies are proposed to investigate how EBNA1 and cellular factors direct replication to initiate at an element of oriP called DS. Four specific aims are described: (1) While a specific arrangement of EBNA1-binding sites at DS is critical to support replication, flanking sequences performan undefined auxiliary role. We will investigate whether auxiliary sequences help EBNA1 to occupy DS in vivo, help to recruit cellular ORC and MCM complexes to DS, and function through an ability to bind cellular proteins TRF1 and TRF2. (2) We will determine precisely where replication initiates at or near DS. If a unique site on one side of DS is preferred, as suggested by preliminary data, we will investigate whether this site is determined by local sequences, by the position of nucleosomes, or by the direction of the FR element of oriP. (3) We will investigate why certain EBNA1 mutants retain DNA-binding activity but are impaired for oriP- specific DMA replication. These mutants should help to determine how EBNA1 proteins interact with host factors and with each other to form a functional replicator at DS. (4) We will test whether transcription through DS after it has been licensed for DMA replication results in the removal of MCM complexes. Because replication licensing cannot occur after cyclin-dependent kinases have been activated at the G1/S border, the question has broader significance for understanding patterns of initiation of DMA replication on the latent EBV chromosome and on human chromosomes.