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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要当非人类适应性毒株战胜人类时，就会出现毁灭性的甲型流感病毒 (IAV) 大流行成功感染和维持人际传播的障碍。大流行毒株的新颖性构成了对人类健康造成沉重负担，导致严重呼吸道并发症的发生率增加，与季节性流感相比，住院率和死亡率更高。 2009 年最近一次大流行 (pH1N1) 源自含有人类、猪和禽类适应基因片段的重配猪 IAV。到要了解这些大流行的 IAV 如何克服物种障碍，我们必须了解其机制导致病毒适应人类宿主。我们实验室最近的工作表明，基因调控 IAV M 片段的表达在宿主适应中发挥作用。 IAV M 段编码两个主要蛋白质通过选择性剪接。矩阵 1 (M1) 赋予病毒体其结构，并由共线 mRNA 编码，病毒复制所需的M2由M1 mRNA的剪接产物编码。我们已经用过反向遗传学来设计重组病毒，这些病毒共享八个 IAV 基因片段中的七个，但在 M 段的起源，使我们能够评估该段的属性。携带重组病毒禽类或人类适应的 M 片段复制到相似的滴度并表达相似水平的 M1 和 M2 蛋白当感染禽类细胞时。然而，在人类细胞中，禽类 M 段病毒的复制效率较低，并且与人类M段病毒相比，M2 mRNA和蛋白质的表达水平明显更高。哺乳动物适应的 M 片段在人体细胞中维持低水平的 M2。这些数据导致我的假设是，引入哺乳动物宿主后，需要对禽类 IAV M 片段进行突变才能重新建立对 M2 表达调节很重要的最佳病毒-宿主相互作用。在目标 1 中，我将绘制地图鸟类和哺乳动物之间 M 基因表达差异的 M 序列决定因素适应M段。我使用反向遗传学生成了携带核苷酸的禽类 M 片段病毒 pH1N1 M 片段中发现的突变。我将用这些病毒感染人类细胞。那我来评价一下这些突变对鸟类和人类适应的 M1 和 M2 mRNA 和蛋白质稳态水平的影响和稳定性。在目标 2 中，我将研究宿主因素在禽类和禽类之间 M2 表达差异中的作用。人类适应的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剪接因子并评估 M1 和 M2 mRNA 的亚细胞定位显微镜下观察感染。通过这些实验，我将阐明其背后的分子机制。物种特异性 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，可用于更好地评估未来 IAV 人畜共患疾病的大流行风险。