The influenza A virus nucleoprotein: structure and function

甲型流感病毒核蛋白：结构和功能

• 批准号: 8133640
• 负责人: Robert M Krug
• 金额: $ 37.29万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8133640
• 关键词: Acute; Affect; Affinity; Anisotropy; Antiviral Agents; Binding; Biological; Biological Assay; Cell Nucleus; Cells; Charge; Complex; Development; Electron Microscopy; Epidemic; Exhibits; Family; Fluorescence Anisotropy; Gene Expression; Genome; Genomics; Grant; Health; Human; In Vitro; Infectious salmon anemia virus; Influenza; Influenza A virus; Influenza B virus; Influenza Virus Infected Cells; Laboratories; Length; Link; Methods; Modeling; Molecular; Molecular Biology; Mutagenesis; Mutation; N-terminal; Nucleoproteins; Orthomyxoviridae; Phosphorylation; Play; Polymerase; Prevention; Process; Proteins; Public Health; RNA; RNA Binding; RNA Viruses; RNA chemical synthesis; RNA replication; Recombinants; Research; Rhabdoviridae; Ribonucleoproteins; Role; Scaffolding Protein; Site; Site-Directed Mutagenesis; Structure; System; Tail; Techniques; Transfection; Viral; Viral Genes; Viral Genome; Virus; Virus Assembly; Virus Diseases; X-Ray Crystallography; drug development; flexibility; influenza A virus nucleoprotein; influenzavirus; monomer; mortality; mutant; pandemic disease; reconstruction; respiratory; three dimensional structure; trafficking; viral RNA

DESCRIPTION (provided by applicant): Influenza A virus causes annual epidemics and is potential threat for widespread pandemics. The genome of influenza A virus consists of eight segments of (-)RNA that are encapsidated in distinct double-helical structures called the ribonucleoprotein (RNP) complexes. The nucleoprotein (NP), the major protein component of RNPs, binds along genomic RNA at a 24nt interval. The viral polymerase, consisting of PA, PB1, and PB2, is bound to the two RNA termini of the RNP. NP is abundantly made in infected cells, and is essential for important viral processes such as viral genome trafficking, viral RNA replication, and virus assembly. Our laboratory has recently determined the crystal structure of influenza A virus NP, which shows an overall fold and an external RNA binding mode different from those of rhabdoviruses. Several key residues for RNA binding have been identified by mutagenesis. Polarization anisotropy assay shows that wild-type NP exhibits strong binding affinity for RNA, which in turn stimulates NP self-oligomerization. Without RNA, NP oligomers are not stable and slowly dissociate to monomers. Mutant NP monomer that cannot oligomerize shows weak RNA binding, suggesting that either RNA binds to NP-NP interface or NP monomer has a tertiary structure different from oligomeric NP. Purified NP can be phosphorylated at S402/S403 in vitro, and phosphorylation reduced NP RNA binding. Using purified recombinant NP and viral polymerase, we have shown that NP stimulates unprimed viral RNA synthesis by interacting directly with the polymerase. Our specific aims are to: 1. Elucidate the linked mechanisms of NP RNA-binding, oligomerization and assembly into RNPs. We will: (1) determine the structure of the NP monomer to elucidate the mechanism of NP monomer?oligomer transition; (2) determine the RNA binding mode of NP using site-directed mutagenesis and fluorescence anisotropy; (3) solve the structure of the NP:RNA complex by X-ray crystallography and cryo-EM reconstruction to obtain an accurate atomic model for the structure of the RNP; (4) elucidate the effects of phosphorylation of specific sites of NP on its various functions; and (5) solve the structure of NP from infectious salmon anemia virus (ISAV) and influenza B virus and determine whether NP self-oligomerization and RNA binding modes are broadly conserved in the Orthomyxoviridae family. 2. Elucidate the role of NP in viral RNA replication. To understand how NP interacts with the polymerase and affects RNA synthesis, we will: (1) identify the polymerase PA sequence(s) that regulate NP-directed unprimed viral RNA replication; (2) identify the sites on NP that functionally interact with the polymerase; and (3) use our in vitro system to elucidate several key issues concerning NP-dependent viral RNA replication. Our proposed research employs a broad spectrum of X-ray crystallography, electron microscopy and other biophysical techniques (by Dr. Tao, PI) together with functional and virological methods (by Dr. Krug, co-PI). Results from our studies will have important applications in antiviral drug development. PUBLIC HEALTH RELEVANCE: Influenza viruses cause highly contagious, acute respiratory illnesses and pose a serious threat to the human public health. The influenza virus nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complex, and plays critical roles in viral RNA replication. The aim of this grant is to elucidate the molecular mechanism and biological significance of several key activities of the influenza A virus nucleoprotein and to discover how such activities can be exploited for antiviral development.

描述（由申请人提供）：甲型流感病毒每年都会引起流行，并且是大流行的潜在威胁。甲型流感病毒的基因组由八个 (-)RNA 片段组成，这些片段被包裹在称为核糖核蛋白 (RNP) 复合物的独特双螺旋结构中。核蛋白 (NP) 是 RNP 的主要蛋白质成分，以 24nt 的间隔沿着基因组 RNA 结合。病毒聚合酶由 PA、PB1 和 PB2 组成，与 RNP 的两个 RNA 末端结合。 NP 在受感染的细胞中大量产生，对于病毒基因组运输、病毒 RNA 复制和病毒组装等重要病毒过程至关重要。我们实验室最近测定了甲型流感病毒NP的晶体结构，其整体折叠和外部RNA结合模式与弹状病毒不同。 RNA 结合的几个关键残基已通过诱变鉴定出来。偏振各向异性测定表明，野生型 NP 对 RNA 表现出很强的结合亲和力，进而刺激 NP 自身寡聚化。没有 RNA，NP 寡聚体不稳定并缓慢解离为单体。不能寡聚化的突变 NP 单体表现出较弱的 RNA 结合，表明 RNA 与 NP-NP 界面结合，或者 NP 单体具有与寡聚 NP 不同的三级结构。纯化的 NP 可在体外在 S402/S403 处磷酸化，磷酸化会减少 NP RNA 结合。使用纯化的重组 NP 和病毒聚合酶，我们发现 NP 通过直接与聚合酶相互作用来刺激未引发的病毒 RNA 合成。我们的具体目标是： 1. 阐明 NP RNA 结合、寡聚化和组装成 RNP 的相关机制。我们将：（1）确定NP单体的结构，阐明NP单体→低聚物转变的机制； (2)利用定点突变和荧光各向异性确定NP的RNA结合模式； (3)通过X射线晶体学和冷冻电镜重建解析NP:RNA复合物的结构，以获得RNP结构的精确原子模型； (4)阐明NP特定位点的磷酸化对其各种功能的影响； (5)解析传染性鲑鱼贫血病毒(ISAV)和乙型流感病毒的NP结构，并确定NP自身寡聚化和RNA结合模式在正粘病毒科中是否广泛保守。 2.阐明NP在病毒RNA复制中的作用。为了了解 NP 如何与聚合酶相互作用并影响 RNA 合成，我们将：（1）识别调节 NP 引导的未引发病毒 RNA 复制的聚合酶 PA 序列； (2) 识别 NP 上与聚合酶功能性相互作用的位点； (3) 使用我们的体外系统阐明有关 NP 依赖性病毒 RNA 复制的几个关键问题。我们提出的研究采用了广泛的X射线晶体学、电子显微镜和其他生物物理技术（由陶博士，PI）以及功能和病毒学方法（由Krug博士，共同PI）。我们的研究结果将在抗病毒药物开发中具有重要应用。公共卫生相关性：流感病毒会引起高度传染性的急性呼吸道疾病，对人类公共卫生构成严重威胁。流感病毒核蛋白形成螺旋基因组核糖核蛋白复合物的蛋白支架，并在病毒RNA复制中发挥关键作用。该资助的目的是阐明甲型流感病毒核蛋白几个关键活性的分子机制和生物学意义，并发现如何利用这些活性进行抗病毒开发。