Influenza nucleoprotein interactions and viral RNA synthesis

流感核蛋白相互作用和病毒RNA合成

• 批准号: 8586319
• 负责人: Laura Lynn Newcomb
• 金额: $ 10.73万
• 依托单位: CALIFORNIA STATE UNIV SAN BERNARDINO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-08 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8586319
• 关键词: Amino Acid Sequence Homology; Amino Acids; Antiviral Agents; Antiviral Therapy; Biochemical; Cell Nucleus; Code; Cytoplasm; Data; Defect; Development; Disease; Electrophoretic Mobility Shift Assay; Foundations; Fright; Gel; Gene Expression; Generations; Genes; Genetic Transcription; Goals; Hand; Health; Human; In Vitro; Influenza; Influenza A virus; Knowledge; Laboratories; Length; Life Cycle Stages; Light; Mission; Molecular; Mutate; N-terminal; Nature; Nucleoproteins; Outcome; PTPN11 gene; Pharmaceutical Preparations; Phenotype; Plasmids; Proteins; Public Health; RNA; RNA Binding; RNA Processing; RNA Viruses; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Recombinants; Research; Role; Severities; Structure; System; Testing; Transfection; Vaccine Production; Viral; Viral Proteins; Virus; Work; arm; defined contribution; design; global health; in vivo; influenza A virus nucleoprotein; influenzavirus; inhibitor/antagonist; innovation; mutant; novel; pandemic disease; research study; resistant strain; tool; viral RNA; viral rescue

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.

描述（由申请人提供）：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、看不见的亚型，这些亚型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，其在调节病毒RNA合成中的作用尚未完全阐明。该研究旨在填补NP在体内病毒RNA合成和加工中的作用方面的知识空白。NP在甲型流感分离株中具有高度同源性，因此针对NP的抗病毒治疗有望对多种甲型流感亚型有效。该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，以促进创新抗病毒疗法的发展。目的是表征两个NP突变体，它们表现出RNA合成缺陷，并被设计成破坏与病毒RNA依赖性RNA聚合酶（RdRP）或宿主RNA加工因子UAP56的相互作用。中心假设是这些相互作用对病毒体内RNA合成很重要。提出的研究的基本原理是这些NP突变体的特征将描述适合作为抗病毒靶点的病毒-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验证：1)表征以破坏与病毒RdRP相互作用为目标的流感NP突变体；2)定义n端NP相互作用的作用