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

基本信息

批准号：
10231509
负责人：
Lea Gaucherand
金额：
$ 4.18万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10231509
关键词：
3-Dimensional Address Adult Respiratory Distress Syndrome Affect Air Animal Model Anti-Inflammatory Agents Antiinflammatory Effect Antiviral Agents Back Binding Bioinformatics Biological 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 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

项目摘要

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，但过多的炎症给宿主带来痛苦，并导致发病率和死亡率。然而，现有的治疗方法很有限。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因此，流感病毒仍然导致数万人死亡。仅在美国，每年就有 50 万人感染流感，而自从流感本身进化以来，全世界就有 50 万人感染流感。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甲型流感病毒调节宿主的新途径的一项策略。对感染的反应部分是通过其病毒编码的核糖核酸酶（RNase）PA-X 事实上，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虽然 PA-X 会全面降解宿主 mRNA，但这种活性如何具体发挥作用。通过转录组分析尚不清楚是否会导致免疫和炎症反应的调节。 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，同时不伤害其他基因，重要的是，PA-X 优先靶向先天免疫基因，这是一致的。凭借其体内抗炎表型，我们的RNAseq数据还发现，剪接的RNA更多。与无内含子 RNA 相比，PA-X 降解的能力更强，我使用报告敏感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，但是这种基于剪接的机制是如何实现的。 PA-X 调节先天免疫和炎症的作用尚不清楚，在拟议的工作中，我将对其进行测试。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 募集到 RNA 中的作用。在目标 2 中，我将探讨特定剪接步骤的作用。 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，I。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 Gaglia实验室的3D肺培养模型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，我们扩大了对分子机制的理解我将通过我的 PA-X 行动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我的导师在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。