Project Summary The goal of this R21 is to develop a high-throughput screen for identification of new small molecule inhibitors for Epstein-Barr virus (EBV) latent infection, and its associated pathologies. EBV is a prevalent human herpesvirus that has been classified by the World Health Organization as a human carcinogen. The latent infection is associated with multiple human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinomas, Hodgkin's lymphoma, gastric carcinomas, and immunoblastic B-cell lymphoma's during immunosuppression. Currently, no therapies exist that target latent infection, and therefore it remains impossible to effectively treat or prevent EBV-associated disease. The latent infection depends on one viral encoded protein, EBNA1, which functions in the replication and maintenance of the viral genome. Genetic and biological disruption of EBNA1, including siRNA depletion and dominant negative mutants, block viral latent infection and EBV-dependent B-cell growth. The EBNA1 DNA binding domain has been characterized structurally and biochemically, and serves as an ideal molecule for targeted small molecule inhibition of EBV infection. We have produced preliminary data that validates EBNA1 as a necessary factor for the maintenance of the EBV viral genome, and have demonstrated that we can synthesize highly pure EBNA1 protein that retains its specific DNA-binding activity, and that this binding activity can be monitored in vitro. In this R21 proposal, we will extend these findings to develop a reliable assay for EBNA1 activity that can be used in HTS format to screen for small molecule inhibitors. The risk of this proposal is substantially mitigated by our previous successful development of a similar assay for another EBV regulatory factor (a lytic, rather than a latent phase protein). Public Health Relevance Statement The significance of this proposal to human health will be the development of new therapies for eliminating EBV latent infection and preventing or treating associated malignant pathologies. Episomal maintenance of EBV genomes during latency is essential for the propagation and survival of EBV. Moreover, tumor cells latently infected with EBV typically harbor episomal genomes, suggesting that episomal maintenance is required for EBV pathogenesis. With the exception of HU, no other pharmacological agents have been shown to effectively eliminate EBV episomes from latently infected cells. However, targeting latent genomes with EBNA1 directed siRNA, dominant negatives, or binding site competitors have been shown to be effective inhibitors of EBV-driven B-cell proliferation and viability. The goals of this application are to identify highly specific inhibitors of EBNA1 DNA binding function. These studies may provide important new reagents for manipulation of EBV latent infection, and may also provide new opportunities to interrupt latent infections of EBV-related viruses, including Kaposi's Sarcoma Associated Herpesvirus (KSHV) and human papillomavirus (HPV).