Replication and maintenance of the Epstein-Barr virus genome in latently infected cells

Epstein-Barr virus (EBV), a member of the herpesvirus family, causes lifelong infections in humans. Approximately 90% of the world's adult population is infected with EBV and although most of these infections are benign, EBV is causally associated with certain human cancers of B-lymphocytes and epithelial cells. The EBV genome, a double-stranded DNA molecule of ~170 kbp, is present within EBV-positive tumor cells as a multicopy plasmid. EBV plasmids are each replicated once per cell cycle during S phase and are efficiently partitioned between daughter cells during mitosis. The best characterized EBV replication origin utilized in latently-infected cells is located adjacent to a sequence element required for plasmid maintenance and, together, these two elements are referred to as oriP. Small recombinant plasmids bearing oriP are also replicated in a cell cycle-regulated manner and the replication and the long-tem maintenance of these oriP-plasmids requires only a single viral protein, the EBV nuclear antigen-1 (EBNA-1). Due to the availability of quantitative short-term replication assays for oriP-plasmids and the almost comlete dependence of oriP-plasmid replication upon cellular proteins, oriP provides an extremely attractive system with which to investigate the regulated initiation of DNA replication from human chromosomal replication origins.

A long-term goal of this laboratory is to obtain a detailed molecular description of the mechanisms by which replication initiates within oriP. EBNA-1 binds site-specifically to four sites within the oriP replicator and bends the DNA but is unable to unwind the duplex in preparation for daughter strand synthesis. Therefore, it is likely that cellular replication proteins carry out this early step in oriP-plasmid DNA replication. We have determined that the oriP replicator contains a core element, consisting of two EBNA-1 binding sites flanked by inverted copies of a 14-bp repeat, that is essential for activity and multiple ancillary elements that are required for full activity. The results of our genetic analysis of the oriP replicator suggest that a host cell protein binds to nucleotides within the 14-bp repeats and interacts with EBNA-1 to promote the initiation of DNA replication. It was recently shown that the human origin recognition complex (ORC), minichromosome maintenance protein complex (MCM), and geminin participate in the regulated replication of plasmids bearing oriP. Our current efforts are directed at identifying the protein and sequence requirements for the physical and functional association of human replication proteins with the replicator of oriP and determining the contribution of ancillary elements to replicator function.