PROJECT SUMMARY Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the etiological agent of several AIDS/HIV-associated human neoplasms, including the B cell malignancy primary effusion lymphoma (PEL). During PEL pathogenesis, KSHV remains predominantly latent, expressing few proteins and approximately 25 mature microRNAs (miRNAs). Despite their identification nearly two decades ago, the roles of the KSHV miRNAs in PEL remain poorly understood, partially due to challenges in achieving lasting miRNA loss-of-function. Specifically, the high copy number of KSHV genomes in PEL precludes CRISPR-based KSHV genome editing and transfectable antisense inhibitors only achieve transient inhibition. My goal is therefore to leverage novel insights into the endogenous miRNA turnover mechanism to define the essentiality of KSHV miRNAs in PEL cell lines. In my preliminary work, I have already improved upon lentivirally-deliverable miRNA inhibitors to achieve stable miRNA inhibition in cultured PEL cells. My preliminary data suggests that at least one KSHV miRNA, miR-K11, a highly expressed KSHV mimic of the oncogenic human miRNA miR-155, is essential for PEL cell viability. My proposal seeks to broaden our understanding of KSHV-encoded miRNAs. My overall hypothesis is that individual KSHV miRNAs promote the survival or proliferation of PEL cells. I further hypothesize that miR-K11 plays a central role in PEL oncogenesis, by repressing specific uncharacterized target mRNAs. To investigate these hypotheses, I propose two Specific Aims. In Specific Aim 1, I will design, validate, and employ lentiviral miRNA inhibitors to determine the importance of each KSHV miRNA for the viability or proliferation of a set of PEL cell lines. For miRNAs with significant essentiality, I will assess their effects on the cellular and viral transcriptomes, KSHV lytic reactivation, apoptosis, and cell cycle progression. In Specific Aim 2, I propose to define the essential role of miR-K11 in PEL. I will first extend my finding that miR-K11 is required for the viability of PEL cells to an extended set of PEL cell lines. I will then use genome- wide and custom CRISPR rescue screens to identify which miR-K11 targets underlie miR-K11 essentiality. I will mine hits from these screens, new RNA-seq experiments, and published KSHV miRNA targetome data sets, to prioritize and validate functionally essential targets of miR-K11. Collectively, my research plan will define the importance of KSHV encoded miRNAs and identify the functionally essential targets of miR-K11 in PEL. Since miR-K11 mimics a cellular miRNA that contributes to human lymphomas, my results will also be broadly relevant to mechanisms of lymphomagenesis, potentially paving the way for new therapeutic strategies. My research plan will finally allow me to gain invaluable skills in molecular biology, bioinformatics, mentoring, and written and oral presentations, helping me reach my goal of running an independent research laboratory at ...