Project Summary/Abstract Kaposi sarcoma (KS) remains one of the most common malignancies in people living with HIV/AIDS worldwide. Kaposi sarcoma herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), is the causal agent for KS. The oral mucosa is the first target of KSHV infection once the virus is in the oral cavity. However, the initial infection process of the mucosa by KSHV has never been studied, mainly due to lack of an in vitro model to recapitulate the viral infection in vivo and nonexistence of an animal model for KSHV infection. We have created the 3- dimentional (3-D) organotypic culture as an oral mucosal mimic resembling a stratified oral mucosa with the potential for histological assessment of the KSHV infection/transmission process. Our studies show that KSHV infection in the 3-D oral mucosa model is enhanced by saliva extracellular vesicles (EVs), particularly exosomes, from HIV patients. Exosomes purified from the saliva of HIV patients or from culture media of HIV-infected T cells contain similar HIV-specific cargos, including HIV TAR RNA. KSHV entry into target cells and its infectivity are increased by HIV-positive saliva exosomes in primary and immortalized human oral epithelial cells. Further, KSHV infection and transmission to the suprabasal cells in the 3-D organotypic culture are enhanced by HIV- positive exosomes. Increased KSHV infectivity by HIV-positive exosomes is attributed to the HIV TAR RNA within the vesicles and epidermal growth factor receptor (EGFR) of host cells. Inhibition of EGFR by Cetuximab, a monoclonal antibody to EGFR, blocks KSHV infection enhanced by HIV-positive exosomes. Taken together, these data lead us to hypothesize that HIV-positive exosomes promote KSHV infection and transmission through the oral route and are responsible for increased incidence of KSHV infection in people living with HIV. To test this hypothesis, we plan to 1) assess KSHV transmission and infection through the oral route with the 3-D cultures of oral mucosal and tonsil tissues incorporated with peripheral blood mononuclear cells (PBMCs) as well as the epithelium-endothelium model. KSHV infection and transmission through the epithelial barrier to immune and endothelial cells in response to HIV+ saliva exosomes will be assessed; 2) delineate the mechanism by which HIV-positive exosomes promote KSHV oral transmission using the 3-D tissue models. We will apply single cell RNAseq to identify cell-specific changes in transcriptome of the oral mucosal and tonsil 3-D tissue models following KSHV infection in response to HIV+ exosomes; and 3) elucidate the role of EGFR-dependent mitogen-activated protein kinase activation in enhanced KSHV transmission by HIV-positive exosomes. Success of the proposed research will advance our understanding of KSHV oral transmission at the molecular level and the underlying mechanisms for developing potential novel therapeutic strategies.