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）：流感病毒会导致年度流行病，并且是广泛流行病的潜在威胁。流感A的基因组由八个（ - ）RNA的片段组成，这些片段被封装在不同的双螺旋结构中，称为核糖核蛋白（RNP）络合物。核蛋白（NP）是RNP的主要蛋白质成分，以24nt的间隔沿基因组RNA结合。由PA，PB1和PB2组成的病毒聚合酶与RNP的两个RNA末端结合。 NP在感染细胞中大量生产，对于重要的病毒过程，例如病毒基因组运输，病毒RNA复制和病毒组装至关重要。我们的实验室最近确定了流感A病毒NP的晶体结构，该晶体结构显示出与色牛病毒不同的整体折叠和外部RNA结合模式。通过诱变已经鉴定出RNA结合的几个关键残基。极化各向异性测定表明，野生型NP对RNA表现出强大的结合亲和力，进而刺激NP自聚物化。没有RNA，NP低聚物不稳定，并且缓慢地与单体分离。无法寡聚的突变NP单体显示出弱的RNA结合，这表明RNA与NP-NP界面或NP单体的结合具有与低聚NP不同的三级结构。纯化的NP可以在体外在S402/S403处磷酸化，磷酸化降低了NP RNA的结合。使用纯化的重组NP和病毒聚合酶，我们表明NP通过与聚合酶直接相互作用来刺激未刺激的病毒RNA合成。我们的具体目的是：1。阐明NP RNA结合，寡聚和组装的链接机制。我们将：（1）确定NP单体的结构，以阐明NP单体？低聚物转变的机理； （2）使用位置定向的诱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EM重建解决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特定位点磷酸化对其各种功能的影响； （5）从传染性的鲑鱼贫血病毒（ISAV）和流感B病毒中求解NP的结构，并确定NP自我结构化和RNA结合模式是否在Orthomyxoviridae家族中广泛保守。 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的合成，我们将：（1）确定调节NP指导的未取代病毒RNA复制的聚合酶PA序列； （2）识别NP与聚合酶相互作用的位点； （3）使用我们的体外系统来阐明有关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X射线晶体学，电子显微镜和其他生物物理技术（由Tao博士，PI）以及功能性和病毒学方法（由Krug博士，Co-Pi）以及其他生物物理技术的广泛谱。我们研究的结果将在抗病毒药物开发中具有重要的应用。公共卫生相关性：流感病毒引起高度传染性，急性呼吸道疾病，并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质支架，并在病毒RNA复制中起关键作用。该赠款的目的是阐明流感A A A型病毒核蛋白的几种关键活性的分子机制和生物学意义，并发现如何利用此类活动进行抗病毒药性发育。