Host factors and viral determinants mediating flavivirus NS1 tissue-specific endothelial dysfunction and vascular leak ABSTRACT The flavivirus (FV) genus contains medically important mosquito-borne human pathogens that cause a major global disease burden. While dengue (DENV), yellow fever (YFV), and Zika (ZIKV) viruses are systemic, and West Nile (WNV), Japanese encephalitis (JEV), and Zika viruses cause neurotropic infections, each FV can cause severe disease characterized in part by endothelial barrier dysfunction – the most classic example being vascular leak in severe dengue. This may result from overproduction of vasoactive cytokines as well as viral factors. The highly conserved FV non-structural protein 1 (NS1) is secreted from infected cells and circulates in the blood of infected humans. We and others have shown that FV NS1 can trigger endothelial barrier disruption in vitro and vascular leak in mice, independently from virus infection. In our current R01, we showed that endo- cytosis of FV NS1 into endothelial cells (ECs) followed by activation of key enzymes such as cathepsin L and heparanase leads to disruption of the endothelial glycocalyx layer (EGL) as well as mislocalization of intercellular junction proteins, both critical for maintaining endothelial barrier integrity. Interestingly, we found that FV NS1 proteins display exquisite tissue tropism, triggering EC dysfunction in vitro and in vivo in a manner reflecting tissue tropism and disease manifestations of each virus. While FV NS1 tissue tropism was determined by differential EC binding and internalization, downstream activation of key enzymes and signaling pathways required for pathogenesis appear to be conserved across FVs. However, host factors, viral determinants, and mechanisms mediating these processes are unknown. We hypothesize that distinct host factors on tissue- specific ECs mediate FV NS1 cell binding and internalization, leading to endothelial barrier dysfunction, virus dissemination, and different FV disease manifestations. In contrast, once a FV NS1 protein is internalized, we hypothesize that downstream steps of EC dysfunction are comparable – thus pointing the way to a pan-FV intervention. Here, we expand our previous work by identifying and characterizing host glycans, proteins, and NS1 determinants required for tissue-specific cell binding and internalization of NS1 in human ECs, mouse models, and clinical samples. We also define common mechanisms by which FV NS1 proteins trigger pathology. In Aim 1, we will identify and characterize host glycans and FV NS1 determinants required for differ- ential binding to tissue-specific ECs. In Aim 2, we will identify proteinaceous NS1 receptors required to initiate EC dysfunction and define mechanisms by which FV NS1 proteins mediate disruption of the EGL and intercellular junctions in tissue-specific ECs in vitro and in vivo. Aim 3 investigates the impact of FV NS1-mediated endothelial dysfunction on FV dissemination a...