Epstein-Barr virus (EBV) is a ubiquitous human gamma-herpesvirus that establishes life-long latency in B- lymphocytes. EBV is also a causative agent of diverse lymphoid and epithelial malignancies. The variety of EBV tumor types can be partly attributed to a plasticity of viral gene expression and DNA replication programs. These different gene expression programs allow the virus to adapt to a wide range of host cell types and differentiation states, to survive environmental stress conditions and evade host innate and adaptive antiviral immunity. How EBV genomes establish stable latent infections capable of adapting to various host environments, and how this promotes cancer cell evolution remains an important, unanswered question. In this proposal, we will test the hypothesis that EBV gene expression and replication programs are coordinately regulated with host cell information through complex epigenetic mechanisms. The assembly of chromatin and the patterning of histone post-translational modifications is an early event in the establishment of latent infection and determining the transcriptional competence of the viral genome. We will continue ongoing investigations into the mechanism of viral chromatin assembly during primary infection. Aim 1 will focus on the interaction of the viral tegument protein BNRF1 with cellular histone chaperone and intrinsic anti-viral factors Daxx-ATRX to generate latent episomes competent for transcription. Aim 2 will investigate how the viral chromatin is organized by architectural factors CTCF and cohesins, that facilitate functional interactions between promoters and enhancers to regulate viral gene expression. We will also determine how CTCF prevents epigenetic drift between chromosome domains. Finally, Aim 3 will determine how viral and host master-regulatory transcription factors function cooperatively with chromatin organizing factors and epigenetic modifiers to establish dynamic gene expression programs that are responsive to changes in host cell conditions, including those associated with carcinogenesis. We will use next-generation genomic methods to investigate how the viral epigenome is configured in different latency types. We will use high-resolution chromosome conformation methods to investigate DNA regulatory interactions that may determine viral gene expression programs. We will also use biochemical and viral genetic methods to experimentally validate and test the mechanistic basis for epigenetic control. The studies will provide important new information on how EBV gene expression programs are coordinated with host-cell biology, and also provide new insights into the epigenetic control of viral genes associated with EBV carcinogenesis.