Influenza nucleoprotein interactions and viral RNA synthesis

描述（由申请方提供）：甲型流感病毒仍然是一种健康威胁，这种分段RNA病毒的快速进化性质导致出现新的、未知的亚型，这些亚型有可能导致病毒大流行。流感病毒核蛋白（NP）是一种多功能的病毒蛋白，其在调节病毒RNA合成中的作用尚未完全阐明。该研究的目的是填补有关NP在体内病毒RNA合成和加工中的作用的知识的差距。NP在甲型流感分离株之间高度同源，因此预期靶向NP的抗病毒疗法对多种甲型流感亚型具有疗效。该研究策略的目的是发现流感RNA合成所需的NP的生物化学相互作用，以确定流感基因表达的分子机制，并确定新的抗病毒靶点，长期目标是刺激创新抗病毒疗法的发展。目的是表征两个NP突变体，其显示RNA合成缺陷，并被设计为破坏与病毒RNA依赖性RNA聚合酶（RdRP）或宿主RNA加工因子UAP 56的相互作用。核心假设是这些相互作用对体内病毒RNA合成很重要。拟议研究的基本原理是，这些NP突变体的表征将描绘适合作为抗病毒靶点的病毒-病毒相互作用。将通过追求以下两个具体目标来检验该中心假设：1）表征靶向破坏与病毒RdRP的相互作用的流感NP突变体;和2）定义N-末端NP相互作用的作用 病毒RNA合成所必需的。为了分析这些NP突变体的RNA合成和加工活性，将采用创新的转染系统来检查来自vRNA（-）和/或cRNA（+）模板的表达。这一创新策略有利于减轻公众对实验室中重组突变流感病毒产生的恐惧，但更重要的是，这一策略将允许鉴定NP突变体，这些NP突变体可能证明对病毒是致命的，因此不会作为重组突变病毒回收。目的1关注NP和RdRP之间的病毒-病毒相互作用。目标2靶向病毒-宿主相互作用，但如初步结果所示，该NP突变体被病毒NS拯救，因此可能定义重要的病毒-病毒相互作用。强有力的初步数据证明了所提出的实验来表征这些NP突变体的可行性。进行免疫纯化和凝胶迁移试验的工具已经准备就绪，并在申请人的实验室中建立为可行的。预期的结果将产生积极的影响，通过识别和表征重要的病毒-病毒相互作用，可能作为新的抗病毒靶点。这项工作将通过定义参与基本病毒-病毒相互作用的NP的氨基酸做出重大贡献，并为开发针对这些NP相互作用的抗病毒治疗奠定基础，并有效对抗多种甲型流感亚型，以控制流感相关疾病。

DESCRIPTION (provided by applicant): Influenza a virus remains a health menace and the rapidly evolving nature of this segmented RNA virus leads to the emergence of new, unseen subtypes, which have potential to cause a viral pandemic. Influenza nucleoprotein (NP) is a multifunctional viral protein whose role in regulating viral RNA synthesis has not been fully elucidated. The aim of the proposed research is to fill the gap in knowledge regarding the role of NP in viral RNA synthesis and processing in vivo. NP is highly homologous among influenza A isolates and thus antiviral therapies targeting NP are expected to have efficacy against multiple influenza A subtypes. The aim of this research strategy is to discover biochemical interactions of NP required for influenza RNA synthesis in order to define molecular mechanisms underlying influenza gene expression and identify novel antiviral targets with the long term goal to spur development of innovative antiviral therapies. The objective is to characterize two NP mutants which display RNA synthesis defects and were designed to disrupt interaction with either the viral RNA dependent RNA polymerase (RdRP) or host RNA processing factor UAP56. The central hypothesis is that these interactions are important for viral RNA synthesis in vivo. The rationale for the proposed research is that characterization of these NP mutants will delineate viral-viral interactions suitable as antiviral targets. This central hypothesis will be tested by pursuing the following two specific aims: 1) Characterize an influenza NP mutant targeted for disruption of interaction with the viral RdRP; and 2) Define the role of N-terminal NP interactions required for viral RNA synthesis. To analyze these NP mutants for RNA synthesis and processing activities, an innovative transfection system will be employed to examine expression from vRNA(-) and/or cRNA(+) templates. This innovative strategy is beneficial to alleviate the public fear associated with generation of recombinant mutant influenza viruses in the laboratory, but more important this strategy will allow identification of NP mutants which might prove lethal to the virus and thus would not be recovered as a recombinant mutant virus. Aim 1 focuses on the viral-viral interaction between NP and RdRP. Aim 2 targeted a viral-host interaction but as shown in the preliminary results, this NP mutant is rescued by viral NS, and will thus likely define important viral-viral interactions. The strong preliminary data demonstrate feasibility of the proposed experiments to characterize these NP mutants. Tools to perform immuno purification and gel shift assays are already on hand and established as feasible in the applicant's laboratory. The expected outcomes will have a positive impact by identifying and characterizing important viral-viral interactions which may serve as novel antiviral targets. The work will make a significant contribution by defining amino acids of NP involved in essential viral-viral interactions and lay the foundation for development of antiviral therapies targeting these NP interactions and effective against multiple influenza A subtypes to control influenza related disease.