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，最著名的是引起寨卡病毒相关的 小头畸形，登革热和西尼罗河热。这项研究旨在阐明分子 黄素非结构蛋白1（NS1）的机理，一种多功能毒力蛋白。 细胞内NS1对于复制病毒RNA基因组至关重要，而分泌的NS1相互作用 具有先天免疫蛋白，在某些情况下会诱导疾病表型。我们的晶体结构 Zika病毒（ZIKV），登革热病毒2（DENV2）和西尼罗河的全长糖基化NS1 病毒（WNV）将指导实验确定哪些NS1的不同领域负责 它的几个功能中的哪一个。尽管它们的总体相似性（〜50％相同的氨基酸序列），但 特定于单个黄素病毒的几种特性归因于NS1蛋白，从而使 比较分析在解剖NS1的分子功能方面特别有力。广泛 基于DENV2和WNV NS1比较诱变的突变体面板将扩展到 包括ZIKV NS1。基于最初比较研究中NS1影响病毒颗粒的观察结果 组装，我们将检验以下假设，即NS1通过协助病毒结构来充当感染因子 蛋白质折叠或病毒颗粒通过分泌系统。细胞内NS1位于 ER管腔作为与膜相关的二聚体，在复制中与病毒的关联在复制中起关键作用 跨膜蛋白。我们将通过诱变和 生物物理实验。电子冷冻微镜或晶体学将用于研究 分泌NS1的结构，一种六聚体脂蛋白颗粒。 NS1重塑的最初观察 膜将在使用荧光标记的光学显微镜的详细实验之后进行详细的实验 脂质以确定此关键关联中的任何脂质偏好。绑定实验将确定哪个 NS1的域与补体系统的两个蛋白质和先天蛋白的域相互作用 免疫收费受体4。结果将为抗病毒药物的发展提供基础 和/或有效的疫苗，这些疫苗是寨卡，登革热或西尼罗河病毒的有限用途。