Molecular Mechanism of Action of the Influenza PA-X Host Shutoff Protein

流感 PA-X 宿主关闭蛋白的分子机制

• 批准号: 9913984
• 负责人: Marta Maria Gaglia
• 金额: $ 39.42万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-25 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9913984
• 关键词: Affect; Antiviral Agents; Avian Influenza; Bacterial Infections; Binding; Biological Assay; Cell Culture Techniques; Cells; Cessation of life; Characteristics; Chickens; Complex; DNA-Directed RNA Polymerase; Data; Disease; Disease Outbreaks; Drug Targeting; Epithelial Cells; Exons; Future; Gene Expression; Gene Silencing; Goals; Immune; Immune response; Immunomodulators; Infection; Inflammatory Response; Inflammatory Response Pathway; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H5N1 Subtype; Influenza A virus; Innate Immune Response; Interferon Type I; Interferons; Knowledge; Label; Length; Link; Lung; Messenger RNA; Modeling; Molecular; Molecular Mechanisms of Action; Morbidity - disease rate; Mus; Outcome; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Play; Polymerase; Positioning Attribute; Process; Proteins; Public Health; RNA; RNA Degradation; RNA Polymerase II; RNA Processing; RNA Splicing; Regulation; Reporter; Research; Ribonucleases; Role; Site; Specificity; Testing; Translations; Variant; Viral; Viral Pneumonia; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; analysis pipeline; base; bronchial epithelium; cellular targeting; cytokine; cytokine release syndrome; experience; experimental study; human pathogen; immunoregulation; improved; in vivo; influenzavirus; innovation; knock-down; lung injury; mRNA Transcript Degradation; mortality; mouse model; mutant; novel; pandemic disease; prevent; protein function; response; secondary infection; success; transcriptome; transcriptome sequencing; viral RNA

Project summary. The innate immune and inflammatory response to influenza A virus is a major contributor to influenza disease, because it promotes viral pneumonia and secondary infections through lung damage. As a consequence, it has recently become clear that blocking influenza virus replication is not sufficient to treat influenza disease. New strategies to block the innate immune and inflammatory responses need to be developed. These strategies could take advantage of the role of viral proteins in modulating host responses, including the host shutoff factor PA-X, which targets host RNAs for degradation. PA-X inhibits immune responses, and viruses that lack PA-X trigger stronger cytokine responses in mouse and chicken infection models. However, these stronger responses do not promote clearance, but in many cases result in increased morbidity and mortality. This indicates that PA-X has a different function from other influenza immune modulators, and that PA-X can be protective for the host. Defining the mechanism of action of PA-X will be important to understand the unique contribution of PA-X to virus-host interplay and to exploit it to modulate host responses to influenza. While host shutoff factors are usually considered indiscriminate, we have found that PA-X is selective, and targets specific types of RNAs. Particularly, we have uncovered a connection between PA-X and splicing of host RNAs. We have found that PA-X preferentially degrades spliced RNAs in infected cells and, through proximity-dependent protein labeling, we have identified candidate interaction partners of PA-X, which are cellular proteins involved in RNA splicing and 3' end processing. These data have led us to the central hypothesis of this project: PA-X selectively targets RNAs through interactions with cellular splicing and RNA processing machinery. The connection between splicing and PA-X activity is novel among host shutoff RNases. It also suggests that through interactions with splicing factors, PA-X may differentially regulate host RNAs with important functions in immune and inflammatory responses, including spliced vs. intronless types of interferons, key antiviral cytokines. Our objective is to examine the connection between PA-X and cellular mRNA splicing and its effects on cytokine regulation. The long-term goal of our research is to link the molecular function of PA-X to its role in vivo. In Aim 1, we will determine how the number of splice sites and their position affects PA-X's ability to target RNAs, and how widely applicable this is to PA-X variants from divergent influenza strains. We will use a combination of reporter assays and high-throughout RNAseq. In Aim 2, we will define the role of candidate cellular co-factors of PA-X in RNA targeting by PA-X, particularly association with target RNAs and subcellular localization of PA-X using knock-down approaches. We will then use viral mutants to test the role of these interactions in mouse infections. We expect that these experiments will define RNA characteristics and proteins that are important for PA-X activity, expanding our understanding of influenza-host interplay and pointing to strategies to alter PA-X activity for immune modulation.

项目摘要。对流感病毒的先天免疫和炎症反应是导致 流感病，因为它通过肺部损伤促进了病毒性肺炎和继发感染。作为 结果，最近很明显，阻止流感病毒复制不足以治疗 流感病。阻止先天免疫和炎症反应的新策略必须是 发达。这些策略可以利用病毒蛋白在调节宿主反应中的作用， 包括主机关闭因子PA-X，该因子靶向宿主RNA进行降解。 PA-X抑制免疫 反应和缺乏PA-X的病毒引发小鼠和鸡感染中的细胞因子反应更强 型号。但是，这些较强的反应不会促进清除率，但在许多情况下会导致增加 发病率和死亡率。这表明PA-X具有与其他流感免疫不同的功能 调节器，该PA-X可以保护主机。定义PA-X的作用机理将是 重要的是要了解PA-X对病毒宿主相互作用的独特贡献并利用它来调节 宿主对流感的反应。虽然宿主关闭因素通常被视为不分青红卷，但我们发现 该PA-X是选择性的，并且针对特定类型的RNA。特别是，我们发现了联系 在PA-X和主机RNA的剪接之间。我们发现PA-X优先降解了剪接的RNA 感染的细胞以及通过邻近性蛋白质标记，我们已经确定了候选相互作用 PA-X的伴侣，它们是参与RNA剪接和3'末端加工的细胞蛋白。这些数据具有 导致我们提出了该项目的中心假设：PA-X通过与细胞的相互作用选择性地靶向RNA 剪接和RNA处理机械。剪接和PA-X活动之间的联系是新颖的 主机关闭RNase。它还表明，通过与剪接因子的相互作用，PA-X可能会差异 调节具有免疫和炎症反应中重要功能的宿主RNA，包括剪接V。 无内在类型的干扰素，关键抗病毒细胞因子。我们的目标是检查 PA-X和细胞mRNA剪接及其对细胞因子调节的影响。我们研究的长期目标是 将PA-X的分子功能与其在体内的作用联系起来。在AIM 1中，我们将确定剪接站点的数量 它们的位置会影响PA-X靶向RNA的能力，以及这是多么广泛地适用于PA-X变体 不同的流感菌株。我们将结合记者测定法和高通量RNASEQ。目标 2，我们将定义PA-X的候选细胞辅助因子在PA-X靶向RNA中的作用，尤其是 与靶RNA和PA-X的亚细胞定位相关联。然后我们会 使用病毒突变体来测试这些相互作用在小鼠感染中的作用。我们希望这些实验 将定义对PA-X活性很重要的RNA特性和蛋白质 流感主持人相互作用，并指出改变PA-X活动以进行免疫调节的策略。