Epstein-Barr virus (EBV) latent infection is associated with several human cancers, including Burkitt's lymphoma, Hodgkin's disease, and nasopharyngeal carcinoma. The latent viral genome exists as a multicopy episome that replicates in synchrony with the cellular chromosomal DNA. Latent cycle DNA replication initiates at OriP and EBNA1 is the only viral protein required for OriP-dependent replication and plasmid maintenance. EBNA1 binds to multiple sites in OriP, but has no intrinsic helicase or other enzyme activity associated with DNA replication function. We have used DNA affinity chromatography to isolate and identify several cellular proteins that associate with OriP in an EBNA1-dependent manner. Our preliminary data indicates that these proteins contribute to plasmid maintenance and the regulation of DNA replication. Several of these proteins have known function at human telomeres, including Telomeric Repeat Binding Factor 2 (TRF2), hRap1, and Tankyrase. TRF2 and hRap1 bind telomeric repeats and regulate chromosome stability. We now show that EBNA1 stimulates TRF2 binding to the nonamer repeats (TTAGGG) in the Dyad symmetry region of OriP. Mutation of the nonamer repeats reduced plasmid maintenance function of OriP and sensitizes OriP to genotoxic stress. We propose that the nonamer-binding proteins function as a DNA damage checkpoint that regulates replication of OriP. Failure to regulate replication leads to a loss of stable plasmid maintenance. However, it is not clear how nonamer-binding proteins execute this function. In this application we propose to determine the structural organization of nonamer binding proteins at OriP. We will determine their protein interactions and their ability to effect single strand formation, subcellular localization, nuclear matrix attachment, and DNA looping between regions of OriP. We have also found that nonamer-binding proteins possess poly-ADP ribose activity, and we will determine how NAD levels and DNA damage may regulate the activity of PARP proteins associated with OriP. We will also determine if EBNA1 is a substrate of PARP in vivo, and if this modification regulates replication or plasmid maintenance function. We will investigate the role of nonamer-binding proteins in modifying OriP DNA and/or chromatin structure. Finally, we will determine if nonamers provide a DNA checkpoint function by arresting OriP replication in response to genotoxic stress. We hypothesize that the nonamer-binding proteins increase stability of the latent viral genome by protecting it from catastrophic recombination and degradation. The experiments proposed in this application will reveal important new insights into the mechanism of EBV latent cycle DNA replication and plasmid maintenance, and may have important implication for other latent herpesviruses, as well as the functions of cellular proteins involved in telomere maintenance.
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