Epstein-Barr Virus (EBV) persists in a latent state in B lymphocytes, where it normally causes limited infected cell proliferation. In immune deficient people, including people with advanced HIV infection, EBV infected cell proliferation is a major cause of Lymphoproliferative Diseases, Lymphoma, and Hodgkin Disease. The long term objective of this program is to develop strategies to prevent, contain, and eliminate harmful EBV infections, particularly infections that result in uncontrolled or partially controlled lymphocyte proliferation. EBV Nuclear Antigens EBNA3A and EBNA3C are each uniquely essential for EBV infected cell proliferation. One essential pathway through which EBNA3A and EBNA3C cause lymphocyte proliferation is through interaction with RBP/CSL, the Notch pathway protein link to gene transcription and oncogenesis. The first specific aim of the program is to identify EBNA3A domains that are critical for cell growth, the important interacting cell proteins, and the pathways through which EBNA3A interacts with RBP/CSL, CtBP, and other critical proteins to alter cell growth and survival. The second specific aim is to identify EBNA3C domains that are critical for cell growth, the important interacting cell proteins, and the pathways through which EBNA3C interactions with RBP/CSL and CtBP alter cell gene transcription, growth, and survival. The third specific aim is to understand the fundamental sub-molecular interactions of EBNA3A and EBNA3C with RBP/CSL in vitro and in vivo, at levels that will better enable recombinant EBV reverse genetic analyses and molecular target development for tool compound screening. New approaches will be taken to understand this interaction at the structural level and to identify interactions with therapeutic potential. The effect of EBNA3A and EBNA3C association with RBP/CSL on EBNA2, NotchIC, and cognate DNA association, in vitro and in LCLs, will be evaluated using tandem affinity (TAP) and sequential affinity (SAP) pulldown and LC/MS. We will determine which cell genes are regulated by EBNA3A and EBNA3C by Chromatin Immunoprecipitation and transcriptional profiling. To better understand the EBNA3A and EBNA3C interactions with RBP/CSL, we will attempt to solve the crystal structure of the RBP/CSL complex in the presence and/or absence of cognate DNA and employ reverse yeast two-hybrid assays. Knowledge of the critical EBNA3A and EBNA3C residues for RBP/CSL interaction will allow us to test whether EBNA3A or EBNA3C polypeptides that have the highest affinity can interrupt LCL growth. Collectively, these experiments will provide a detailed picture of how the EBV nuclear proteins subvert and precisely regulate components of Notch signaling and other pathways. PUBLIC HEALTH RELEVANCE Epstein-Barr Virus infected cell proliferation is a major cause of malignant Lymphoproliferative Diseases, Lymphoma, and Hodgkin's Disease in immune deficient people, especially people with advanced HIV infection. The objective of this program is to develop strategies to prevent, contain, and eliminate harmful EBV infections that result in uncontrolled or partially controlled lymphocyte proliferation. This proposal is to identify the essential roles of two virus encoded nuclear proteins in infected cell proliferation and to identify targets for interrupting their effects.