Summary The Epstein Barr virus (EBV) is a causal agent in Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, gastric cancer, nasopharyngeal carcinoma and autoimmune diseases. In the context of HIV co-infection (+ or - ART), EBV is especially problematic with a substantially elevated risk of EBV-associated lymphomas. The overall objective of this P01 is to investigate the role of non-coding RNAs in HIV-associated malignancies. Our assessment of EBV-associated Burkitt’s lymphoma (BL) and gastric cancer (GC) patient datasets revealed that outside of the ubiquitously expressed small EBER1/2 non-coding viral RNAs, in vivo viral gene expression is primarily limited to two overlapping long non-coding RNAs, A73 and RPMS1 and two large miRNA clusters contained within the RPMS1 introns. Strikingly, we found that viral miRNAs comprise up to 75% of all miRNAs expressed in the cell in both BL and GC clinical samples. MiRNA targetome analyses revealed that EBV (and KSHV (Project 1)) miRNAs interact more effectively with their targets than their cellular counterparts, in part through targeting more accessible regions of RNAs and through forming hybrids with more favorable binding energies. Functionally, we found that EBV miRNAs likely suppress innate and adaptive immune responses to viral infection. Together, these studies support the contention that EBV miRNAs are key contributors to the tumor phenotype in EBV associated lymphomas. Although certain cell miRNAs are known oncogenes, there is extensive evidence for a generalized lower expression of cell miRNAs in cancer. Further, driver mutations in miRNA processing genes have been identified in a number of cancer types. Utilizing patient RNA-seq data, we found that cell miRNA processing is diminished in EBV positive tumors and we show that expression of EBV miRNA clusters in EBV negative cells causes decreased processing of endogenous cell miRNAs. We hypothesize that the introduction of multiple copies of ectopic high-density miRNA clusters into cells upon EBV infection not only yields high viral miRNA production but also causes sequestration of microprocessor resources and inhibited processing of cell miRNAs. We further hypothesize that viral microprocessor inhibition diminishes the expression of cell tumor suppressor miRNAs to influence the tumor phenotype in a manner similar to genetic alterations of miRNA processing factor genes. In this proposal, we will use bench work, viral recombineering (Core C), and bioinformatics (Core B) in tissue culture, animal models (Core D), and clinical models to 1) determine the functional impact of direct viral miRNA targeting on immune regulatory pathways and 2) delineate the underlying mechanisms, the impacted oncogenic pathways, and the functional consequence of EBV-mediated microprocessor overload on the tumor phenotype. We will also begin preliminary studies to determine whether microprocessor overload is a shared feature extending to other oncogenic herpesviruses through testi...