Japanese encephalitis virus (JEV) is a mosquito-transmitted flavivirus that is closely related to other emerging viral pathogens, including dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout Southeast Asia, the Pacific Islands, and the Far East, causing around 68,000 cases of infection worldwide each year. In this report, we present a 2.1-Å-resolution crystal structure of the C-terminal β-ladder domain of JEV nonstructural protein 1 (NS1-C). The surface charge distribution of JEV NS1-C is similar to those of WNV and ZIKV but differs from that of DENV. Analysis of the JEV NS1-C structure, with in silico molecular dynamics simulation and experimental solution small-angle X-ray scattering, indicates extensive loop flexibility on the exterior of the protein. This, together with the surface charge distribution, indicates that flexibility influences the protein-protein interactions that govern pathogenicity. These factors also affect the interaction of NS1 with the 22NS1 monoclonal antibody, which is protective against West Nile virus infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 mediates interaction with membranes and that sulfate binding sites common to NS1 structures are not glycosaminoglycan binding interfaces. This report highlights several differences between flavivirus NS1 proteins and contributes to our understanding of their structure-pathogenic function relationships.

IMPORTANCE JEV is a major cause of viral encephalitis in Asia. Despite extensive vaccination, epidemics still occur. Nonstructural protein 1 (NS1) plays a role in viral replication, and, because it is secreted, it can exhibit a wide range of interactions with host proteins. NS1 sequence and protein folds are conserved within the Flavivirus genus, but variations in NS1 protein-protein interactions among viruses likely contribute to differences in pathogenesis. Here, we compared characteristics of the C-terminal β-ladder domain of NS1 between flaviviruses, including surface charge, loop flexibility, epitope cross-reactivity, membrane adherence, and glycosaminoglycan binding. These structural features are central to NS1 functionality and may provide insight into the development of diagnostic tests and therapeutics.

日本脑炎病毒（JEV）是一种通过蚊子传播的黄病毒，与其他新出现的病毒病原体密切相关，包括登革热病毒（DENV）、西尼罗河病毒（WNV）和寨卡病毒（ZIKV）。乙脑病毒感染可导致脑膜炎和脑炎，严重时会导致永久性脑损伤和死亡。JEV 主要发生在东南亚、太平洋岛屿和远东的农村地区，每年在全球造成约 68,000 例感染病例。在本报告中，我们展示了 JEV 非结构蛋白 1 (NS1-C) C 端 β-梯结构域的 2.1 Å 分辨率晶体结构。JEV NS1-C 的表面电荷分布与 WNV 和 ZIKV 相似，但与 DENV 不同。通过计算机分子动力学模拟和实验解决方案小角度 X 射线散射对 JEV NS1-C 结构进行分析，表明该蛋白质外部具有广泛的环灵活性。这与表面电荷分布一起表明灵活性影响控制致病性的蛋白质-蛋白质相互作用。这些因素也会影响 NS1 与 22NS1 单克隆抗体的相互作用，后者可预防西尼罗河病毒感染。脂质体和肝素结合测定表明，只有 NS1 的 N 末端区域介导与膜的相互作用，并且 NS1 结构共有的硫酸盐结合位点不是糖胺聚糖结合界面。该报告强调了黄病毒 NS1 蛋白之间的几个差异，有助于我们理解它们的结构-致病功能关系。

重要性JEV 是亚洲病毒性脑炎的主要原因。尽管广泛接种疫苗，流行病仍然发生。非结构蛋白 1 (NS1) 在病毒复制中发挥作用，并且由于它是分泌性的，因此可以与宿主蛋白表现出广泛的相互作用。NS1 序列和蛋白质折叠在黄病毒属内是保守的，但病毒之间 NS1 蛋白质-蛋白质相互作用的变化可能导致发病机制的差异。在这里，我们比较了黄病毒之间 NS1 的 C 端 β-梯结构域的特征，包括表面电荷、环灵活性、表位交叉反应性、膜粘附和糖胺聚糖结合。这些结构特征是 NS1 功能的核心，可以为诊断测试和治疗的发展提供深入的见解。