Host factors and viral determinants mediating flavivirus NS1 tissue-specific endothelial dysfunction and vascular leak

宿主因子和病毒决定簇介导黄病毒NS 1组织特异性内皮功能障碍， 血管渗漏 摘要 黄病毒（FV）属包含医学上重要的蚊媒人类病原体，其引起主要的 全球疾病负担。虽然登革热（DENV）、黄热病（YFV）和寨卡（ZIKV）病毒是全身性的， 西尼罗河病毒（WNV）、日本脑炎病毒（JEV）和寨卡病毒引起嗜神经性感染，每种FV都可以 导致严重的疾病，部分特征是内皮屏障功能障碍-最经典的例子是 严重登革热的血管渗漏。这可能是由于血管活性细胞因子以及病毒性血管炎的过度产生。 因素高度保守的FV非结构蛋白1（NS 1）由感染细胞分泌并在细胞中循环 感染者的血液我们和其他人已经表明，FV NS 1可以触发内皮屏障破坏， 体外和小鼠血管渗漏，与病毒感染无关。在我们目前的R 01中，我们显示了endo- FV NS 1胞吞进入内皮细胞（EC），随后激活关键酶如组织蛋白酶L和 乙酰肝素酶导致内皮糖萼层（EGL）的破坏以及细胞间 连接蛋白，两者都是维持内皮屏障完整性的关键。有趣的是，我们发现FV NS 1 蛋白质显示出精致的组织向性，在体外和体内触发EC功能障碍， 每种病毒的组织嗜性和疾病表现。而FV NS 1的组织嗜性是通过 差异EC结合和内化，下游关键酶和信号通路的激活 发病机制所需的基因似乎在FV中是保守的。然而，宿主因素、病毒决定因子和 介导这些过程的机制是未知的。我们假设组织上不同的宿主因子- 特异性EC介导FV NS 1细胞结合和内化，导致内皮屏障功能障碍，病毒 传播和不同的FV疾病表现。相反，一旦FV NS 1蛋白被内化， 假设EC功能障碍下游步骤是可比较的-从而指出了泛FV的途径 干预在这里，我们通过鉴定和表征宿主聚糖、蛋白质， 人内皮细胞、小鼠内皮细胞和小鼠内皮细胞中组织特异性细胞结合和NS 1内化所需的NS 1和NS 1决定簇 模型和临床样本。我们还定义了FV NS 1蛋白触发 病理在目标1中，我们将鉴定和表征不同宿主所需的聚糖和FV NS 1决定簇。 与组织特异性EC的基本结合。在目标2中，我们将鉴定启动免疫应答所需的蛋白质类NS 1受体。 EC功能障碍和定义FV NS 1蛋白介导EGL和细胞间干扰的机制 在体外和体内的组织特异性EC的连接。目的3研究FV NS 1介导的内皮细胞凋亡对细胞增殖的影响。 功能障碍对FV传播和发病机制的影响 越南、尼加拉瓜和巴西的登革热和YF患者。这项工作得到了糖生物学、FV 结构生物学和生物化学，血管生物学，FV发病机制和动物模型，以及临床 研究和应该确定严重FV疾病的生物标志物和新的病毒和宿主治疗靶点。

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 and pathogenesis in mouse models and human clinical samples from severe dengue and YF patients in Vietnam, Nicaragua and Brazil. This work is supported by experts in glycobiology, FV structural biology and biochemistry, vascular biology, FV pathogenesis and animal models, and clinical investigation and should identify biomarkers of severe FV disease and novel viral and host therapeutic targets.