Epstein-Barr Virus (EBV) reprograms host cell gene expression and metabolism during the establishment of latency and the immortalization of B-lymphocytes. The regulatory mechanisms coordinating this reprogramming with EBV latency reflect important events in viral oncogenesis, yet remain poorly understood. We have found that key viral regulators of EBV latency, including the major tegument protein BNRF1 and the EBV Nuclear Antigen EBNA1 coordinate key aspects of purine metabolism during establishment of latency and immortalization of primary B-cells. In this R01, we focus on how EBV reprograms purine metabolic gene expression, and how purine metabolites contribute directly to EBV tumorigenesis. One clue to this coordinate regulation is provided by the viral-encoded tegument protein BNRF1 that shares extensive structural similarity to the purine biosynthetic enzyme FGARAT (also called PFAS) and functions in viral chromatin assembly during primary infection. Orthologues of BNRF1 are found in all gamma herpesviruses, including KSHV ORF75, and share the common function of disarming components of the PML-nuclear body (PML-NB) and its anti-viral functions. We have previously shown that BNRF1 interacts with the histone H3.3 chaperone DAXX and displaces its interaction with the ATP-dependent SNF2-like helicase ATRX to enable selective expression of latency-specific viral genes during primary infection. However, it has not yet been shown how the viral FGARAT homology domain is linked to cellular purine biosynthesis and/or signaling. Using metabolomics mass spectrometry, we provide new preliminary data indicating that the purine biosynthetic pathway is among the most significantly perturbed by EBV during B-cell immortalization. Integrating gene expression (RNA-Seq), chromatin accessibility (ATAC-Seq), and EBNA1-DNA binding to host chromosome (ChIP-Seq), we identified cellular metabolic genes, including adenine deaminase (ADA), adenosine kinase 4 (AK4), and purinergic receptors P2RY8 and P2RX5 as direct targets of EBNA1 transcriptional regulation during EBV immortalization. We now propose to investigate the mechanisms by which EBV senses and reprograms purine metabolism and how purinergic signaling regulates establishment of EBV latency and host cell transformation. We will test the central hypothesis that EBV coordinately regulates cellular purine metabolism with viral and cellular gene expression during the B-cell immortalization process, and that purinergic signaling is critical for viral latency and oncogenesis. Specifically, we will investigate how EBV regulates expression of purine metabolic genes during primary infection (Aim 1), elucidate how purine metabolism impacts the establishment of EBV latency (Aim 2), and investigate the role of purine metabolism and signaling in B-cell immortalization, immune signaling, and EBV-induced tumorigenesis (Aim 3). These studies will advance our understanding of basic mechanisms coordinating gene expression with me...