pproximately 250,000 infants acquire human immunodeficiency virus (HIV-1) infection annually despite antiretroviral prophylaxis, suggesting the need for alternative prevention strategies. Mother-to-child transmission (MTCT) is an important pathway for the spread of HIV from mother to fetus and infant; however, the molecular mechanisms of HIV MTCT are poorly understood. Our recent work showed that >90% of virions internalized into infant tonsil epithelium are sequestered in the endosomes, including multivesicular bodies (MVBs) and vacuoles of epithelial cells, for up to 9 days. In contrast, such prolonged intracellular sequestration of HIV was not observed in adult tonsil epithelial cells: intracellular virus was rapidly inactivated. This is consistent with our published work showing that intracellular HIV is inactivated in adult polarized epithelial cells by high-level expression of anti-HIV innate immune proteins human beta defensin 2 (HBD2) and HBD3. However, fetal and infant oral epithelial cells did not express HBDs, leading to transmission of infectious HIV. In ongoing experiments we have found that the interaction of herpes simplex virus-1 (HSV-1), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV) with the apical (mucosal) surface of infant tonsil epithelial cells containing HIV-1 leads to the exocytosis of HIV virions, which are infectious for peripheral blood mononuclear cells (PBMCs). HSV, HCMV, and EBV are oral microbiota of infants, and their interaction with infant tonsil epithelial cells containing HIV may lead to the release and spread of HIV into CD4+ T lymphocytes, macrophages, and Langerhans/dendritic cells (LCs/DCs), leading to HIV MTCT. Virtually nothing is known about the role of herpesvirus oral microbiota in HIV MTCT. Therefore, investigation of the molecular mechanisms underlying HSV-, HCMV-, and EBV-associated HIV spread from neonatal/infant oral epithelia into HIV-susceptible cells will undoubtedly lead to a better understanding of the oral microbiota-associated pathogenesis of HIV MTCT and the development of a new preventive therapeutic strategy against perinatal viral infection. Accordingly, the specific aims of this proposal are (1) To study the molecular mechanisms of HSV-, HCMV-, and EBV-induced HIV exocytosis in infant tonsil epithelium containing sequestered HIV; (2) To investigate the role of HSV, HCMV, and EBV in HIV MTCT through disruption of mucosal epithelium and activation of HIV-susceptible cells; and (3) To study the potential mechanisms of intravesicular HIV inactivation by antiviral innate immune proteins human beta defensin 2 (HBD2) and HBD3. This work will greatly advance knowledge of the role of herpesvirus oral microbiota in the pathogenesis of HIV MTCT. The proposed studies may lead to the establishment of new therapeutic approaches based on inhibition or reduction of herpesvirus- associated HIV spread from mucosal epithelial cells into HIV target cells and elimination of intraepithelial reservoirs, which in turn may reduce HIV MTCT.