Virus-host interactions underlying species specificity of influenza A virus gene expression

项目摘要 当非人类适应的毒株战胜人类物种时，毁灭性的甲型流感病毒（IAV）大流行就会出现 成功感染和维持人际传播的障碍。一种新奇的大流行病毒株 对人类健康造成巨大负担，导致严重呼吸道并发症的发病率增加， 与季节性流感相比，住院和死亡。2009年最新的大流行（pH 1 N1） 来源于含有人、猪和禽类适应性基因片段的重组猪IAV。到 为了了解这些大流行性IAV如何克服物种障碍，我们必须了解 导致病毒适应人类宿主。我们实验室最近的工作表明， IAV M片段的表达在宿主适应中起作用。IAV M片段编码两个主要的 选择性剪接的蛋白质。基质1（M1）赋予病毒体其结构并由共线mRNA编码， 而病毒复制所需的M2由M1 mRNA的剪接产物编码。我们已经使用 反向遗传学工程重组病毒，共享八个IAV基因片段中的七个，但不同的是， M段的起源，使我们能够评估该段的属性。重组病毒携带 禽或人适应的M片段复制到相似的滴度并表达相似水平的M1和M2蛋白 感染鸟类细胞的时候。然而，在人类细胞中，禽类M节段病毒复制效率较低， 表达比人M节段病毒显著更高水平的M2 mRNA和蛋白。哺乳动物 适应的M片段在人细胞中维持低水平的M2。这些数据让我产生了一个假设， 在引入哺乳动物宿主后，需要对禽类IAV M片段进行突变以重新 建立对M2表达调节重要的最佳病毒-宿主相互作用。在目标1中，我将映射 鸟类和哺乳动物M基因差异表达M序列决定簇 适应M段。我用反向遗传学方法制造了携带核苷酸的M片段病毒 在pH 1 N1 M片段中鉴定的突变。我将用这些病毒感染人类细胞。然后我会评估 这些突变对禽类和人类适应性M1和M2 mRNA和蛋白质稳态水平的影响 与稳定在目标2中，我将研究宿主因素在禽类和哺乳动物间M2表达差异中的作用。 人类适应的M片段。我将确定鸟类和人类M1 mRNA与宿主的结合亲和力， 剪接因子，并评估M1和M2 mRNA的亚细胞定位， 显微镜下的感染。通过这些实验，我将阐明的分子机制， 种特异性IAV M片段基因表达。这些研究将揭示IAV的其他机制 对人类宿主的适应性，可用于更好地评估未来IAV人畜共患病的大流行风险。

PROJECT SUMMARY Devastating influenza A virus (IAV) pandemics arise when non-human adapted strains overcome human species barriers to successfully infect and sustain human-to-human transmission. The novelty of a pandemic strain poses a substantial burden to human health leading to increased incidences of severe respiratory complications, hospitalizations, and death when compared to seasonal influenza. The most recent pandemic in 2009 (pH1N1) arose from a reassortant swine IAV that contained human, swine, and avian adapted gene segments. To understand how these pandemic IAVs overcome species barriers, we must understand the mechanisms that lead to viral adaptation to human hosts. Recent work in our lab has demonstrated that regulation of gene expression from the IAV M segment plays a role in host adaptation. The IAV M segment encodes two major proteins by alternative splicing. Matrix 1 (M1) gives the virion its structure and is encoded by the colinear mRNA, and M2, which is needed for viral replication, is encoded by a splice product of the M1 mRNA. We have used reverse genetics to engineer recombinant viruses that share seven of the eight IAV gene segments but differ in the origin of the M segment, allowing us to evaluate properties of this segment. Recombinant viruses carrying avian or human adapted M segments replicate to similar titers and express similar levels of M1 and M2 protein when infecting avian cells. However, in human cells, the avian M segment virus replicates less efficiently and expresses significantly higher levels of M2 mRNA and protein than the human M segment virus. Mammalian adapted M segments maintain low levels of M2 in human cells. These data lead to my hypothesis that, following introduction into mammalian hosts, mutations to the avian IAV M segment are needed to re- establish optimal virus-host interactions important for regulation of M2 expression. In Aim 1, I will map the M sequence determinants that underly differential M gene expression between avian and mammalian adapted M segments. I have used reverse genetics to generate avian M segment viruses that carry nucleotide mutations identified in the pH1N1 M segment. I will infect human cells with these viruses. Then I will evaluate the impact of these mutations on avian and human adapted M1 and M2 mRNA and protein steady state levels and stability. In Aim 2, I will investigate the role of host factors in differential M2 expression between avian and human adapted M segments. I will determine the binding affinities of avian and human M1 mRNAs with host splicing factors using biochemical methods and evaluate subcellular localization of M1 and M2 mRNAs during infection by microscopy. Through these experiments, I will elucidate the molecular mechanisms that underly the species specificity IAV M segment gene expression. These studies will reveal additional mechanisms of IAV adaptation to human hosts, which could be used to better assess the pandemic risks of future IAV zoonoses.