Abstract: Kaposi’s Sarcoma (KS)-Associated Herpesvirus (KSHV) is a human g-herpesvirus responsible for KS, pleural effusion lymphoma (PEL), multlicentric Castleman’s disease (MCD) and KSHV inflammatory cytokine syndrome (KICS). KS and PEL are HIV-AIDS-defining malignancies that occur at high frequency during AIDS, but also after treatment with ART especially in endemic regions. KSHV oncogenesis is driven by the complex life cycle of KSHV involving long-term latent infection of B-lymphocytes, reactivation and persistent infection of lymphatic endothelial and mesenchymal stem cells, and immune evasion. KSHV encoded nuclear antigen LANA is expressed in all KSHV-associated tumors and is essential for maintenance of the viral episome during latent infection in proliferating cells. KSHV LANA is also thought to contribute directly to viral oncogenesis and pathogenesis through interactions with tumor suppressor proteins and rewiring of host gene expression programs. We have previously shown that LANA contributes to the viral DNA replication, epigenetic regulation and higher-order structure of KSHV episomes. We have also identified chromatin organizing factors, such as CTCF and cohesins, as key regulatory factors in the control of latent gene expression and restriction of lytic reactivation. More recently, we have explored KS tumor transcriptomics and drug sensitivity assays to identify metabolic pathways implicated in the regulation of LANA and KSHV-infected cell survival. We now propose to advance each of these findings and continue our long-term effort to understand the role of LANA in episome maintenance, latency regulation, and oncogenesis. Specifically, we will investigate (1) the role of LANA in regulating latency-associated DNA replication and genome integrity through a potential intrinsic tyrosine recombinase activity that regulates replication termination and genome decatenation at the viral terminal repeats, (2) the role of LANA and cellular chromatin organizing factors DAXX, CTCF and cohesin in forming higher-order nuclear bodies that protect and organize the viral episome to maintain stable genomes and gene expression patterns in latently infected cells, and (3) how onco-metabolic stress disrupts LANA function and KSHV latency to drive tumorigenesis. Together these studies provide an integrated framework to further advance our knowledge of KSHV infection and latency, and provide new opportunities for therapeutic intervention in KSHV- associated disease.