Abstract: Epstein-Barr Virus (EBV) a human gammaherpesvirus that is associated with diverse lymphoid and epithelial malignancies. Like all cancers, EBV-associated cancers must compete and survive in nutrient depleted environments. Adaptation to oxygen fluctuation is a hallmark of most cancers. In this proposal, we investigate how oxygen metabolism plays a central role in EBV latency and tumorigenesis. For aim 1, we will investigate how EBV modulates the host hypoxic response during primary infection. Our preliminary data indicates EBNA1 and EBNA2 can bind and regulate cellular genes controlling the hypoxic response, including hypoxia inducible factor 1 alpha (HIF1A). We will test the hypothesis that EBV infection modulates the host response to hypoxia to promote infected cell survival and promote tumorigenesis. For aim 2, we will investigate how the viral epigenome is regulated by oxygen sensing enzymes. EBV associated Burkitt lymphoma (BL) and epithelial malignancies are known to acquire high DNA methylation commonly referred to as the CpG island methylator phenotype (CIMP). We will investigate how DNA methylation is regulated through the oxygen-sensitive control of TET2 enzymes and explore how hypoxia contributes to CIMP and silencing of tumor suppressor genes. For aim 3, we will test how oxygen sensing enzymes regulate EBNA1 and EBV episome maintenance during latency. We have found that proline and lysine hydroxylases regulate EBNA1 and EBNA2 protein stability and function. We have mapped interaction sites on EBNA1 that bind the lysine hydroxylase PLOD1 and found this interaction essential for EBNA1 protein stability under normoxia. We now propose to investigate how hypoxia regulates the interaction of PLOD1 with EBNA1 to control EBV episome maintenance and persistence during latent infection. These aims are highly integrated with other members of the program project exploring hypoxia effects on related tumor viruses. Together, these aims will provide a framework to understand how viruses respond to hypoxia to promote host cell survival and drive the oncogenic process.