RNA targeting specificity and immunomodulation by the influenza A virus ribonuclease PA-X

甲型流感病毒核糖核酸酶 PA-X 的 RNA 靶向特异性和免疫调节

• 批准号: 10493119
• 负责人: Lea Gaucherand
• 金额: $ 2.27万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10493119
• 关键词: 3-Dimensional; Acute Respiratory Distress Syndrome; Address; Affect; Air; Animal Model; Anti-Inflammatory Agents; Antiinflammatory Effect; Antiviral Agents; Back; Binding; Bioinformatics; Cells; Chromatin; Data; Data Set; Development; Disease; Engineering; Environment; Epithelial; Epithelial Cells; Gene Expression; Genes; Genetic Transcription; Goals; Health; High-Throughput Nucleotide Sequencing; Human; Immune; Immune Targeting; Immune response; Immunological Models; Immunoprecipitation; Infection; Inflammation; Inflammatory; Inflammatory Response; Influenza; Influenza A virus; Innate Immune Response; Interferon Type I; Interferons; Introns; Knowledge; Label; Lead; Life; Link; Liquid substance; Lung; Measures; Mentors; Messenger RNA; Modeling; Molecular; Molecular Mechanisms of Action; Morbidity - disease rate; Mus; Mutate; Natural Immunity; Pancreatic ribonuclease; Pathway interactions; Persons; Phenotype; Publications; RNA; RNA Degradation; RNA I; RNA Polymerase II; RNA Splicing; Regulation; Reporter; Ribonucleases; Role; Running; Scientist; Seasons; Specificity; System; Techniques; Testing; Therapeutic; Therapeutic Intervention; Thiouridine; Time; Training; Vaccines; Viral; Viral Proteins; Virus; Virus Diseases; Work; antiviral immunity; base; bronchial epithelium; career; chromatin immunoprecipitation; cytokine; design; experience; experimental study; fighting; immunoregulation; in vivo; influenza infection; influenzavirus; innovation; laboratory experience; lung injury; mortality; novel therapeutics; pandemic disease; prevent; recruit; response; secondary infection; skills; small molecule inhibitor; transcriptome; transcriptome sequencing; transcriptomics; viral transmission; virology; virus host interaction

PROJECT SUMMARY/ABSTRACT Although inflammation is needed for the host to defend itself against influenza infection, too much inflammation is detrimental to the host, and contributes to morbidity and mortality. Yet, available therapeutics are solely antiviral and do not prevent inflammation-driven lung damage, mostly because how inflammation is regulated during influenza infection is not fully understood. As a result, influenza virus still kills tens of thousands of people every year in the US alone, and up to half a million worldwide. Since influenza itself has evolved mechanisms to regulate the host innate immune and inflammatory response, studying these mechanisms is one strategy to start designing new avenues of therapeutic intervention. Influenza A virus modulates host responses to infection in part through its virus-encoded ribonuclease (RNase) PA-X. Indeed, mutated PA-X- deficient viruses cause higher levels of inflammatory responses and increased mortality compared to wild-type viruses in animal models of infection. While PA-X globally degrades host mRNAs, how this activity specifically leads to modulation of the immune and inflammatory response is not known. Through transcriptomic analysis of infected and PA-X expressing cells, the Gaglia lab has found that PA-X actually targets specific subsets of RNAs, while sparing others. Importantly, innate immune genes are preferentially targeted by PA-X, consistent with its in vivo anti-inflammatory phenotype. Our RNAseq data also uncovered that spliced RNAs are more susceptible to PA-X degradation than intronless RNAs, a specificity that I confirmed using reporter constructs, suggesting a mechanistic link between PA-X and splicing. However, how this splicing based mechanism allows PA-X to modulate innate immunity and inflammation is unknown. In the proposed work, I will test the hypothesis that PA-X exploits RNA splicing to target nascent RNAs, allowing PA-X to down-regulate genes that are induced transcriptionally during infection and modulate the host innate immune response and inflammation. In Aim 1, I will study the role of specific splicing steps in recruiting PA-X to RNAs. In Aim 2, I will explore the link between PA-X targeting and transcription, and study the preferential targeting of nascent RNAs. In Aim 3, I will connect these findings to regulation of innate immunity and inflammation by PA-X in a biologically relevant 3D lung culture model. The Gaglia lab provides the best training environment for me to complete this work, as shown by my recent first-author publication, which expanded our understanding of the molecular mechanism of action of PA-X. I will acquire the technical and conceptual skills that are required for this project through my mentor’s comprehensive knowledge of viral control of host gene expression and high-throughput sequencing dataset analysis, my co-mentor’s extensive experience in RNA work and transcription, and our collaborators’ expertise in human primary bronchial epithelial cultures and bioinformatic analysis. My mentors will also help me develop as a scientist to achieve my career goal of running my own virology lab and train young scientists.

项目概要/摘要 虽然宿主需要炎症来防御流感感染，但过多的炎症 对宿主有害，并导致发病率和死亡率。然而，现有的治疗方法仅 抗病毒且不能预防炎症引起的肺部损伤，主要是因为炎症是如何调节的 流感感染期间的情况尚不完全清楚。结果，流感病毒仍然夺去了数万人的生命。 仅在美国每年就有 50 万人，而全世界就有 ​​50 万人。由于流感本身已经进化 调节宿主先天免疫和炎症反应的机制，研究这些机制是 开始设计新的治疗干预途径的一项策略。甲型流感病毒调节宿主 对感染的反应部分是通过病毒编码的核糖核酸酶 (RNase) PA-X 进行的。确实，突变的 PA-X- 与野生型相比，缺陷病毒会导致更高水平的炎症反应和更高的死亡率 感染动物模型中的病毒。虽然 PA-X 会全局降解宿主 mRNA，但这种活性具体如何 是否会导致免疫和炎症反应的调节尚不清楚。通过转录组分析 Gaglia 实验室发现，PA-X 实际上针对受感染细胞和表达 PA-X 的细胞的特定子集 RNA，同时不伤害其他RNA。重要的是，PA-X 优先靶向先天免疫基因，这与 及其体内抗炎表型。我们的 RNAseq 数据还发现，剪接的 RNA 更 比无内含子 RNA 更容易被 PA-X 降解，我使用报告构建体证实了这一特异性， 表明 PA-X 和剪接之间存在机械联系。然而，这种基于剪接的机制如何允许 PA-X 调节先天免疫和炎症的作用尚不清楚。在提议的工作中，我将测试 假设 PA-X 利用 RNA 剪接来靶向新生 RNA，从而允许 PA-X 下调基因 在感染过程中转录诱导并调节宿主先天免疫反应和炎症。 在目标 1 中，我将研究特定剪接步骤在将 PA-X 招募到 RNA 中的作用。在目标 2 中，我将探索 PA-X 靶向和转录之间的联系，并研究新生 RNA 的优先靶向。在目标 3 中，我 将这些发现与 PA-X 在生物学相关的先天免疫和炎症调节中联系起来 3D 肺培养模型。 Gaglia实验室为我完成这项工作提供了最好的训练环境，因为 我最近发表的第一作者出版物表明，它扩展了我们对分子机制的理解 PA-X 的作用。我将通过我的工作获得该项目所需的技术和概念技能 导师对宿主基因表达的病毒控制和高通量测序的全面知识 数据集分析、我的合作导师在 RNA 工作和转录方面的丰富经验以及我们的合作者的 人类原代支气管上皮培养和生物信息学分析方面的专业知识。我的导师也会提供帮助 我发展成为一名科学家，以实现经营自己的病毒学实验室和培训年轻科学家的职业目标。