Abstract: Opportunistic infections are a major cause of morbidity and mortality of AIDS patients in developing countries and AIDS patients are susceptible to a number of cancers caused by opportunistic infections. Kaposi's Sarcoma (KS) is the most common tumor of AIDS patients and is the among the most commonly reported tumor overall in countries in sub-Saharan Africa, where it commonly occurs in both HIV+ and HIV- people. The etiologic agent of KS is Kaposi's Sarcoma-associated herpesvirus (KSHV or HHV-8). KSHV is invariably found in the main KS tumor cell, the spindle cell, where the virus is present predominantly in the latent state. Spindle cells express markers of lymphatic endothelial cells and appear to be most closely aligned with this cell type. While KSHV is clearly an oncogenic virus, little is known about the early steps of how KSHV infection of endothelial cells leads to tumors. While there have been studies that have examined KSHV Induced immortalization and transformation, there is a lack of tractable and reproducible relevant human cell system. We found that KSHV infection of primary neonatal lymphatic endothelial cells (LECs) leads to bypass of senescence, the first step in immortalization. We also demonstrated that the viral Cyclin homolog (vCyc) was essential for KSHV driven proliferation past senescence. KSHV infected neonatal blood endothelial cells (BECs) senesced at the same passage as uninfected blood or lymphatic endothelial cells indicating this phenotype is LEC specific. There is not a strong interferon response in LECs following KSHV infection. In preliminary data we found that knockout of STING in the BECs, preventing the IFN response, made them susceptible to KSHV induced proliferation past senescence, indicating that STING or STING activated pathways prevent KSHV induced proliferation past senescence. In this proposal we will determine if STING expression is sufficient for this phenotype and the mechanism of STING inhibition of KSHV induced proliferation past senescence. We will also determine the mechanism of vCyc activation of endothelial cells to proliferate past senescence. These data provide a robust system for analyzing the mechanism of how KSHV drives the first step of oncogenesis in endothelial cells and why it is specific to LECs. We will further study KSHV induced oncogenesis in our new endothelial precursor cell (EPC) model. We previously found that EPCs isolated from human blood could be separated into lymphatic like EPCs and blood like EPCs. We found that the Ly-EPCs were able to proliferate to low levels in soft agar but uninfected Ly-EPCs and uninfected or KSHV infected Bl-EPCs could not. This presents a second oncogenic assay for studying how KSHV activates cell proliferation in lymphatic endothelial cells but not blood. Ultimately, the proposed studies will help identify how KSHV alters endothelial cells to become cancer cells and ultimately KS tumors and help in the identification of novel therapeu...