Influenza nucleoprotein interactions and viral RNA synthesis

描述(申请人提供)：甲型流感病毒仍然是一个健康威胁，这种节段式RNA病毒的快速进化性质导致了新的、看不见的亚型的出现，有可能导致病毒大流行。流感核蛋白(NP)是一种多功能病毒蛋白，其调节病毒RNA合成的作用尚未完全阐明。这项拟议的研究的目的是填补关于NP在体内病毒RNA合成和处理中的作用的知识空白。NP在甲型流感病毒分离株中同源性很高，因此针对NP的抗病毒治疗有望对多种甲型流感有效。这一研究策略的目的是发现流感RNA合成所需的NP的生化相互作用，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。目的是鉴定两个显示RNA合成缺陷的NP突变体，它们被设计成中断与病毒RNA依赖的RNA聚合酶(RdRP)或宿主RNA处理因子UAP56的相互作用。中心假设是，这些相互作用对体内病毒RNA的合成很重要。这项拟议研究的基本原理是，对这些NP突变体的表征将描绘出适合作为抗病毒靶标的病毒-病毒相互作用。这一中心假设将通过追求以下两个具体目标来检验：1)表征以中断与病毒RdRP相互作用为目标的流感NP突变体；以及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，将使用一种创新的转染系统来检测vRNA(-)和/或cRNA(+)模板的表达。这一创新策略有利于减轻公众对在实验室中产生重组突变流感病毒的恐惧，但更重要的是，这一策略将允许识别可能对病毒致命的NP突变，从而无法恢复为重组突变病毒。目的1研究NP和RdRP之间的病毒-病毒相互作用。目的2以病毒-宿主相互作用为靶标，但初步结果表明，该NP突变体是由病毒NS拯救的，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议的实验的可行性，以表征这些NP突变体。进行免疫纯化和凝胶漂移分析的工具已经准备就绪，并在申请人的实验室中被确定为可行的。预期结果将通过确定和描述重要的病毒-病毒相互作用产生积极影响，这些病毒-病毒相互作用可能成为新的抗病毒靶标。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相互作用的抗病毒疗法和有效对抗多种甲型流感病毒亚型以控制流感相关疾病奠定基础。

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.