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

项目概要/摘要 虽然宿主需要炎症来保护自己免受流感感染，但过多的炎症 对宿主有害，并导致发病率和死亡率。然而，现有的治疗方法仅仅是 抗病毒，不能防止炎症驱动的肺损伤，主要是因为炎症是如何调节的 流感病毒感染的原因尚不完全清楚。因此，流感病毒仍然导致成千上万的人死亡， 仅在美国每年就有50万人死亡，全世界也有50万人死亡。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 一个开始设计新的治疗干预途径的策略。甲型流感病毒调节宿主 对感染的反应部分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，缺陷型病毒引起更高水平的炎症反应和增加的死亡率 感染动物模型中的病毒。虽然PA-X全面降解宿主mRNA，但这种活性如何特异性地 导致免疫和炎症反应的调节是未知的。通过转录组分析 感染和表达PA-X的细胞，Gaglia实验室发现PA-X实际上靶向特定的细胞亚群， RNA，同时保留其他。重要的是，先天免疫基因优先被PA-X靶向，这与PA-X靶向免疫基因一致。 具有体内抗炎表型。我们的RNAseq数据还发现，剪接的RNA更多地 比无内含子的RNA更容易被PA-X降解，这是我用报告基因构建体证实的特异性， 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。在建议的工作中，我将测试 假设PA-X利用RNA剪接靶向新生RNA，使PA-X下调基因， 在感染期间被转录诱导并调节宿主先天免疫应答和炎症。 在目标1中，我将研究特定剪接步骤在招募PA-X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对先天免疫和炎症的调节联系起来， 三维肺培养模型。Gaglia实验室为我完成这项工作提供了最好的培训环境， 我最近的第一作者出版物显示，这扩大了我们对分子机制的理解， PA-X的作用。我将获得的技术和概念的技能，是通过我的这个项目所需的 导师在病毒控制宿主基因表达和高通量测序方面的全面知识 数据集分析，我的共同导师在RNA工作和转录方面的丰富经验，以及我们的合作者 在人类原代支气管上皮细胞培养和生物信息学分析方面的专业知识。我的导师也会帮助我 我发展成为一名科学家，以实现我的职业目标，经营自己的病毒学实验室，培养年轻的科学家。

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.