A Novel RNA Recognition Site on the Influenza B Virus NS1 Protein

乙型流感病毒 NS1 蛋白的新 RNA 识别位点

• 批准号: 8991480
• 负责人: GAETANO T MONTELIONE
• 金额: $ 26.82万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8991480
• 关键词: Accounting; Affect; Affinity; Amides; Antiviral Agents; Apoptosis; Applications Grants; Area; Attenuated; Attenuated Live Virus Vaccine; Binding; Binding Sites; Biological; Biological Assay; C-terminal; Capsid Proteins; Cells; Cessation of life; Characteristics; Chemicals; Circular Dichroism; Code; Collaborations; Complex; DNA; Data; Deuterium; Development; Dimerization; Double-Stranded RNA; Epidemic; Epitopes; Fluorescence Polarization; Future; Gel Chromatography; Genes; Genome; Goals; Health; Hospitalization; Human; Hydrogen; IRF3 gene; Immune response; Infection; Influenza; Influenza A virus; Influenza B Virus; Interferon-beta; Interferons; Laboratories; Ligase; Lung diseases; Maps; Modeling; Molecular; Mutagenesis; Mutation; N-terminal; Nucleic Acid Binding; Nucleic Acids; Oligonucleotides; Pathway interactions; Pattern; Process; Property; Protein C; Proteins; RNA; RNA Binding; RNA Recognition Motif; Recombinants; Research; Research Personnel; Research Project Grants; Ribonucleases; Roentgen Rays; Role; Scanning; Site; Spectrum Analysis; Structure; Surface; Testing; Texas; United States; Universities; Viral; Virulent; Virus; Virus Diseases; Virus Replication; X-Ray Crystallography; austin; base; biophysical properties; biophysical techniques; crosslink; dimer; drug discovery; economic impact; flexibility; flu; influenza epidemic; influenzavirus; insight; interfacial; light scattering; molecular recognition; mortality; novel; nucleic acid binding protein; pathogen; prevent; productivity loss; programs; promoter; protein kinase R; seasonal influenza; sensor; three dimensional structure; vaccine development

 DESCRIPTION (provided by applicant): Influenza viruses cause a highly contagious respiratory disease in humans. Influenza virus types A and B are distinguished by antigenic differences between their viral coat proteins. Influenza B viruses account for 30 - 40% of seasonal epidemics and are responsible for hundreds of thousands of deaths and countless loss of productivity worldwide each year. The long-term goal of this research program is to elucidate the mechanism(s) by which the influenza B non-structural protein 1 (NS1B) suppresses host innate immune response. One function of NS1B is to suppress interferon-ß (IFN-ß) synthesis in virus-infected cells. This activity has been mapped independently to each of its two domains, NS1B- NTD and NS1B-CTD. While there have been extensive structure-function studies of the NS1B-NTD, the mechanism by which the NS1B-CTD independently antagonizes activation of IFN expression is not known. Our laboratory has discovered that the NS1B-CTD is an RNA-binding domain. This totally unanticipated discovery is based on our 2.0-Å X-ray crystal structure of NS1B-CTD. Dimerization of NS1B-CTD creates a broad, strongly-conserved, basic surface, characteristic of nucleic-acid binding proteins. Fluorescence polarization (FP) studies demonstrate that NS1B-CTD binds RNA and (more weakly) DNA. Our specific hypothesis is that NS1B-CTD functions by binding dsRNA molecules, to suppress the innate immune response elicited by viral dsRNA. If this hypothesis is correct, disruption of these protein-nucleic acid interactions in virus-infected cells would provide new insights into the mechanisms of innate immune response, and provide the basis for the development of novel antiviral drugs and live attenuated vaccines. The aims of our R21 proposal are to (i) define the sequence and structural features of RNA required for binding to NS1B-CTD, and (ii) define the structural basis for molecular recognition between NS1B-CTD and RNA molecules. Biophysical methods, including FP spectroscopy, will be used to compare binding of NS1B-CTD to various synthetic RNA and DNA molecules. Ala scanning mutagenesis will be used to identify key protein residues contributing to RNA binding affinity. NMR, chemical crosslinking, amide hydrogen-deuterium exchange, and SAXS data will be combined to characterize the RNA-binding surface epitope of NS1B-CTD and to model the 3D structure of NS1B-CTD:RNA complexes. Efforts will also be made to determine 3D structures of these complexes by X-ray crystallography. The structural and biophysical information generated with this R21 Exploratory Research Grant will provide the starting point for a future multi-investigator R01 proposal directed to understanding the role and mechanism of NS1B-CTD in suppressing innate immune response in flu-infected cells.

 描述（由适用提供）：流感病毒在人类中引起高度传染性的呼吸道疾病。 A和B类型的流感病毒以其病毒外套蛋白之间的抗原差异而区分。流感B病毒占季节性发作的30-40％，每年在全球造成数十万人死亡和无数生产率丧失。该研究计划的长期目标是阐明影响力b非结构蛋白1（NS1B）抑制宿主先天免疫响应的机制。 NS1B的一个功能是抑制病毒感染细胞中的干扰素 - ß（IFN-ß）合成。该活动已独立于其两个域NS1B-NTD和NS1B-CTD映射。尽管NS1B-NTD进行了广泛的结构功能研究，但NS1B-CTD独立拮抗IFN表达激活的机制尚不清楚。我们的实验室发现NS1B-CTD是一个RNA结合域。这一完全意外的发现基于我们NS1B-CTD的2.0-ÅX射线晶体结构。 NS1B-CTD的二聚化产生了核酸结合蛋白的特征广泛，强烈的基本表面。荧光偏振（FP）研究表明，NS1B-CTD结合RNA和（更弱）DNA。我们的具体假设是，NS1B-CTD通过结合dsRNA分子来抑制病毒dsRNA引起的先天免疫响应。如果该假设是正确的，那么在感染病毒的细胞中这些蛋白核酸相互作用的破坏将为您提供有关先天免疫反应机制的新见解，并为开发新型抗病毒药药的发展提供了基础。我们R21建议的目的是（i）定义与NS1B-CTD结合所需的RNA的序列和结构特征，以及（ii）定义NS1B-CTD和RNA分子之间分子识别的结构基础。包括FP光谱在内的生物物理方法将用于比较NS1B-CTD与各种合成RNA和DNA分子的结合。 ALA扫描诱变将用于鉴定有助于RNA结合亲和力的关键蛋白残基。 NMR，化学交联，酰胺氢 - 偏见和SAXS数据将组合以表征NS1B-CTD的RNA结合表面表位，并模拟NS1B-CTD的3D结构：RNA复合物。还将努力通过X射线晶体学确定这些配合物的3D结构。这项R21探索性研究赠款生成的结构和生物物理信息将为未来的多投期R01提案提供起点，该建议旨在理解NS1B-CTD在抑制流感感染细胞中先天免疫反应中的作用和机制。