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

 描述（由申请方提供）：流感病毒引起人类高度传染性呼吸道疾病。A型和B型流感病毒的区别在于它们的病毒外壳蛋白之间的抗原差异。流感B病毒占季节性流行病的30 - 40%，每年导致全球数十万人死亡和无数生产力损失。本研究计划的长期目标是阐明流感病毒B非结构蛋白1（NS 1 B）抑制宿主先天免疫应答的机制。NS 1B的一个功能是抑制病毒感染细胞中干扰素-γ（IFN-γ）的合成。该活性已被独立地映射到其两个结构域NS 1B-NTD和NS 1B-CTD中的每一个。虽然已经对NS 1B-NTD进行了广泛的结构-功能研究，但NS 1B-CTD独立拮抗IFN表达活化的机制尚不清楚。我们的实验室已经发现NS 1B-CTD是一个RNA结合结构域。这一完全出乎意料的发现是基于我们的NS 1B-CTD的2.0-X射线晶体结构。NS 1B-CTD的二聚化产生了广泛的、高度保守的碱性表面，这是核酸结合蛋白的特征。荧光偏振（FP）研究表明，NS 1B-CTD结合RNA和（更弱）DNA。我们的具体假设是NS 1B-CTD通过结合双链RNA分子来抑制病毒双链RNA引发的先天免疫反应。如果这一假设是正确的，这些蛋白质-核酸相互作用在病毒感染的细胞中的破坏将提供新的见解先天免疫反应的机制，并提供新的抗病毒药物和减毒活疫苗的开发基础。我们的R21提案的目的是（i）定义与NS 1B-CTD结合所需的RNA的序列和结构特征，以及（ii）定义NS 1B-CTD和RNA分子之间分子识别的结构基础。生物物理方法，包括FP光谱，将用于比较NS 1B-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质残基。将结合NMR、化学交联、酰胺氢-氘交换和SAXS数据来表征NS 1B-CTD的RNA结合表面表位，并对NS 1B-CTD：RNA复合物的3D结构进行建模。还将努力通过X射线晶体学确定这些复合物的3D结构。该R21探索性研究资助产生的结构和生物物理信息将为未来的多研究者R 01提案提供起点，该提案旨在了解NS 1B-CTD在抑制流感感染细胞先天免疫反应中的作用和机制。

 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.