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

甲型流感病毒基因表达的物种特异性背后的病毒-宿主相互作用

• 批准号: 10221465
• 负责人: Gabrielle K Delima
• 金额: $ 4.6万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221465
• 关键词: Address; Affinity; Alternative Splicing; Amino Acid Sequence; Animals; Avian Influenza A Virus; Binding; Biochemical; Birds; Cell Nucleus; Cells; Cessation of life; Chicken Cells; Complement; Cytoplasm; Data; Domestic Fowls; Electrophoretic Mobility Shift Assay; Engineering; Family suidae; Fluorescent in Situ Hybridization; Future; Gene Expression; Gene Expression Regulation; Genes; Goals; Half-Life; Health; Hospitalization; Human; In Vitro; Incidence; Infection; Influenza A virus; Integration Host Factors; Lead; Life Cycle Stages; Literature; M2 protein; Mammalian Cell; Maps; Mediating; Messenger RNA; Methods; Microscopy; Molecular; Mutation; Nuclear; Nucleotides; Phenotype; Plaque Assay; Play; Prevalence; Production; Property; Proteins; Protons; Public Health; RNA; RNA Splicing; RNA Viruses; Regulation; Research; Risk; Role; Sampling; Series; Shapes; Species Specificity; Structure; Testing; Time; Transcript; Translations; United States; Viral; Viral Genome; Viral reservoir; Virion; Virus; Virus Diseases; Virus Replication; Work; Zoonoses; aquatic bird; base; design; experimental study; fitness; flu transmission; mutant; novel; pandemic disease; pandemic influenza; protein expression; recombinant virus; respiratory; reverse genetics; seasonal influenza; swine influenza; transmission process; viral fitness; virus host interaction

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.

项目摘要 当非人类适应性菌株克服人类时，毁灭性的流感病毒（IAV）流感出现 成功感染和维持人类对人类传播的障碍。大流行菌株的新颖性 对人类健康的重大负担，导致严重呼吸并发症的发生率增加， 与季节性流感相比，住院和死亡。 2009年的最新大流行（PH1N1） 源自包含人，猪和鸟类改编的基因段的重新排化的猪IAV。到 了解这些大流行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。我们实验室的最新工作表明基因的调节 来自IAV M段的表达在宿主适应中起作用。 IAV M段编码两个主要 蛋白质通过替代剪接。矩阵1（M1）赋予病毒粒子的结构，并由Colinear mRNA编码， 病毒复制所需的M2由M1 mRNA的剪接产物编码。我们已经使用过 反向遗传学到工程师重组病毒，共享八个IAV基因段中的七个，但在 M段的起源，使我们能够评估该细分市场的属性。重组病毒携带 鸟类或人类改编的M段复制到相似的滴度，并表达相似水平的M1和M2蛋白 当感染禽类细胞时。但是，在人类细胞中，禽类病毒的复制效率较低，并且 与人类M段病毒相比，M2 mRNA和蛋白质的水平明显高。哺乳动物 改编的M段在人类细胞中保持低水平的M2。这些数据导致了我的假设， 在引入哺乳动物宿主之后，需要对鸟类IAV M段进行突变以重新进行 建立对于调节M2表达很重要的最佳病毒宿主相互作用。在AIM 1中，我将映射 M序列决定因素，即禽和哺乳动物之间的基因表达基因表达 改编的M细分市场。我已经使用了反向遗传学来生成携带核苷酸的鸟类M节病毒 在PH1N1 M段中鉴定出的突变。我将用这些病毒感染人类细胞。然后我会评估 这些突变对鸟类和人类适应的M1和M2 mRNA以及蛋白质稳态水平的影响 和稳定性。在AIM 2中，我将调查宿主因素在鸟类和 人类改编的M段。我将确定鸟类和人类m1 mRNA与宿主的结合亲和力 使用生化方法的剪接因子，并评估M1和M2 mRNA的亚细胞定位 通过显微镜感染。通过这些实验，我将阐明其基础的分子机制 物种特异性IAV M节基因表达。这些研究将揭示IAV的其他机制 适应人类宿主，可以用来更好地评估未来IAV人畜共患病的大流行风险。