Molecular Functions of NS1 Virulence Protein from Dengue and Zika Viruses

登革热和寨卡病毒 NS1 毒力蛋白的分子功能

• 批准号: 9542638
• 负责人: Richard J. Kuhn
• 金额: $ 67.39万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-16 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9542638
• 关键词: Affect; Amino Acid Sequence; Antiviral Agents; Binding; Biological; Biological Assay; Biophysics; Blood Vessels; Cell physiology; Cell surface; Cells; Chimera organism; Cholesterol; Comparative Study; Complement; Complex; Cryoelectron Microscopy; Crystallization; Dangerousness; Dengue; Dengue Virus; Development; Dimerization; Disease; Electron Microscopy; Endocytosis; Extravasation; Flavivirus; Foundations; Genome; HIV; Human; Immune system; Individual; Innate Immune System; Insecta; Integral Membrane Protein; Length; Life Cycle Stages; Lipids; Lipoproteins; Liposomes; Membrane; Membrane Lipids; Membrane Microdomains; Microcephaly; Molecular; Molecular Probes; Mutagenesis; Natural Immunity; Nonstructural Protein; Phenotype; Polysaccharides; Property; Proteins; Public Health; RNA; RNA replication; Reagent; Recombinants; Role; Series; Serotyping; Site; Structure; Surface; System; TLR4 gene; Testing; Tropism; Unspecified or Sulfate Ion Sulfates; Vaccines; Viral; Viral Proteins; Viral Structural Proteins; Virion; Virulence; Virus; Virus Replication; Vitronectin; West Nile Fever; West Nile virus; Zika Virus; base; comparative; complement system; dimer; disease phenotype; electron crystallography; experimental study; inhibitor/antagonist; light microscopy; membrane model; mutant; novel; particle; pathogen; preference; protein folding; receptor; synergism; three dimensional structure

Project Summary Flaviviruses are insect-transmitted human pathogens that most notably cause Zika-associated microcephaly, dengue fever and West Nile fever. This study aims to elucidate the molecular mechanisms of the flaviviral nonstructural protein 1 (NS1), a multi-functional virulence protein. Intracellular NS1 is essential to replication of the viral RNA genome, whereas secreted NS1 interacts with innate immunity proteins and, in some cases, induces disease phenotypes. Our crystal structures of full-length, glycosylated NS1 from Zika virus (ZIKV), dengue virus serotype 2 (DENV2) and West Nile virus (WNV) will guide experiments to identify which of the distinct domains of NS1 are responsible for which of its several functions. Despite their overall similarity (~50% identical amino acid sequences), several properties specific to individual flaviviruses are attributed to the NS1 proteins, making comparative analysis especially powerful in dissecting the molecular functions of NS1. An extensive panel of mutants based on comparative mutagenesis of DENV2 and WNV NS1 will be expanded to include ZIKV NS1. Based on observations in the initial comparative study that NS1 affects virus particle assembly, we will test the hypothesis that NS1 acts as an infectivity factor by aiding viral structural protein folding or virus particle transit through the secretory system. Intracellular NS1 is localized to the ER lumen as a membrane-associated dimer with a critical role in replication through association with viral transmembrane proteins. We will probe the interaction with viral protein NS4B through mutagenesis and biophysical experiments. Electron cryo-microscopy or crystallography will be used to investigate the structure of secreted NS1, a hexameric lipo-protein particle. An initial observation that NS1 remodels membranes will be followed by detailed experiments using light microscopy with fluorescently tagged lipids to determine any lipid preference in this key association. Binding experiments will identify which domains of NS1 interact with which domains of two proteins of the complement system and the innate immunity Toll-like receptor 4. The results will provide a foundation for development of antiviral drugs and/or effective vaccines, which are not available or of limited use for Zika, dengue or West Nile viruses.

项目摘要 黄病毒是昆虫传播的人类病原体，最明显的是引起寨卡病毒相关的 小头症、登革热和西尼罗热。这项研究的目的是阐明分子 黄病毒非结构蛋白1(NS1)，一种多功能毒力蛋白的机制。 细胞内的NS1对病毒RNA基因组的复制是必不可少的，而分泌的NS1则相互作用 具有先天免疫蛋白，在某些情况下，会导致疾病表型。我们的晶体结构 寨卡病毒(ZIKV)、登革2型病毒(DENV2)和西尼罗河病毒的全长糖基化NS1 病毒(WNV)将指导实验，以确定NS1的哪些不同结构域负责 它的几个功能中的哪一个。尽管它们总体上相似(~50%相同的氨基酸序列)， 个别黄病毒特有的几个特性归因于NS1蛋白，使 比较分析在剖析NS1的分子功能方面尤其强大。广泛的 基于DENV2和WNV NS1的比较突变的突变小组将扩大到 包括ZIKV NS1。根据初步比较研究中的观察，NS1影响病毒颗粒 ，我们将测试NS1通过帮助病毒结构作为感染性因子的假设 蛋白质折叠或病毒颗粒通过分泌系统运输。细胞内NS1定位于 内质网作为膜相关二聚体，通过与病毒结合在复制中起关键作用 跨膜蛋白。我们将通过诱变和研究NS4B与病毒蛋白的相互作用 生物物理实验。将使用电子冷冻显微镜或结晶学来研究 分泌的NS1，一种六聚体脂蛋白颗粒的结构。NS1重塑的初步观察 膜之后将使用带有荧光标记的光学显微镜进行详细的实验 脂类来确定这个关键关联中的任何脂类偏好。绑定实验将确定哪些 NS1的结构域与补体系统和固有的两个蛋白质的哪些结构域相互作用 免疫Toll样受体4。这一结果将为抗病毒药物的开发提供基础 和/或对寨卡、登革热或西尼罗河病毒没有或使用有限的有效疫苗。