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

项目总结。对甲型流感病毒的先天免疫和炎症反应是导致 流感疾病，因为它通过肺部损伤促进病毒性肺炎和继发性感染。作为一名 结果，最近变得明显的是，阻止流感病毒复制不足以治疗 流行性感冒。阻断先天免疫和炎症反应的新策略需要 发展起来的。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，它针对主机RNA进行降级。PA-X抑制免疫 在小鼠和鸡的感染中，缺乏PA-X的病毒会触发更强的细胞因子反应 模特们。然而，这些更强烈的反应不会促进清除，但在许多情况下会导致增加 发病率和死亡率。这表明PA-X具有不同于其他流感免疫的功能 调制器，并且PA-X可以保护主机。定义PA-X的作用机制将是 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。虽然主机关闭因素通常被认为是不加区别的，但我们发现 PA-X是有选择性的，并针对特定类型的RNA。特别是，我们发现了一种联系 PA-X和宿主RNA的剪接之间的关系。我们发现PA-X优先降解剪接的RNA。 感染细胞，并通过邻近依赖的蛋白标记，我们已经确定了候选相互作用 PA-X的伙伴，它是参与RNA剪接和3‘端加工的细胞蛋白。这些数据有 这让我们得出了这个项目的中心假设：PA-X通过与细胞相互作用选择性地靶向RNA 剪接和RNA加工机械。剪接和PA-X活性之间的联系是新的 主机关闭RNase。这也表明，通过与剪接因子的相互作用，PA-X可能具有不同的 调节宿主RNA在免疫和炎症反应中具有重要功能，包括剪接vs. 无内含子类型的干扰素，关键的抗病毒细胞因子。我们的目标是研究两者之间的联系 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。在目标1中，我们将确定剪接位点的数量如何 它们的位置影响PA-X靶向RNA的能力，以及这对PA-X变体的广泛适用性 不同的流感病毒株。我们将结合使用记者分析和高通量RNAseq。在AIM 2、我们将明确PA-X的候选细胞辅助因子在PA-X靶向RNA中的作用，特别是 与靶RNA的关联和PA-X的亚细胞定位。到时候我们会的 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。我们预计这些实验 将定义对PA-X活性重要的RNA特征和蛋白质，扩大我们对 并指出了改变PA-X活性以进行免疫调节的策略。

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.