Mechanism for anti-interferon functions of influenza virus

流感病毒抗干扰素功能机制

基本信息

批准号：
10634738
负责人：
Yuan Shi
金额：
$ 39万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-20 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10634738
关键词：
Amino Acids Antigens Antiviral Therapy Area Attenuated Binding Biological CRISPR/Cas technology Cells Co-Immunoprecipitations Data Development Disease Disease Outbreaks Dose Epithelial Cells Exons Ferrets Genes Genome Genomics Goals Hospitalization Host Defense Human Immune Evasion Immunization Programs In Vitro Influenza Influenza A virus Influenza vaccination Interferon Type I Interferons Invaded Knockout Mice Lung Mass Spectrum Analysis Membrane Proteins Messenger RNA Molecular Mus Mutagenesis Mutate Mutation National Institute of Allergy and Infectious Disease Nature Pathogenesis Pathway interactions Pattern Phenotype Play Polymerase Population Positioning Attribute Production Proteins Proteomics Research Role SCID Mice Safety Series Signal Pathway Site Strategic Planning Surface System Testing Vaccination Vaccines Viral Viral Genome Viral Nonstructural Proteins Viral Physiology Viral Proteins Virus Virus Replication Wild Type Mouse adaptive immune response cDNA Library deep sequencing density improved in vivo influenza infection influenza virus vaccine influenzavirus interferon antagonist knockout gene mouse model mutant novel pandemic disease pathogen pathogenic virus protein function protein protein interaction response screening universal influenza vaccine universal vaccine virus host interaction

项目摘要

Project Summary Influenza A virus causes disease in 5-20% of the population with over 200,000 hospitalizations annually in US. The antigen drift and shift of influenza virus pose a serious challenge for flu vaccination program. Although IFN response is known as the first important host defending mechanism to control influenza virus replication at different steps, influenza virus has acquired many strategies to suppress/antagonize IFN function effectively allowing its replication in the hosts. It is well known that influenza viral nonstructural protein 1 antagonizes IFN function through suppressing IFN production. However, less is known on how influenza virus counteracts function of IFN-stimulated genes (ISGs), many of which have antiviral functions. Our recently developed a quantitative and high-throughput genomics system has enabled the identification of novel IFN-sensitive mutations on multiple viral segments across influenza genome. The goal of this project is to determine how influenza virus antagonizes host anti-viral mechanism by studying recently identified novel IFN-sensitive mutations using a comprehensive genomic and proteomic platform in vitro and in vivo. The research plan focuses on three motifs on influenza viral proteins PA, PB1, and M1. Each motif, represented by multiple mutations located together in a small surface area, might target a cellular protein or step of IFNR signaling pathway. In addition, our preliminary data suggest mutations on the M1 motif interact with IFN production pathway, while motifs on PA and PB1 may counteract ISGs downstream of IFN production. The hypothesis is that these three anti-IFN motifs of influenza PA, PB1, and M1 interact with cellular proteins to antagonize IFN functions. A series of complementary approaches including high-density mutagenesis, mRNA display-deep sequencing, ISG cDNA library screening, CRISPR/Cas9 gene editing, human lung primary culture system, and gene knockout mice are proposed in three independent while logically connected Specific Aims (SAs) to test our scientific hypothesis. SA#1 is to define the motifs on viral proteins PA, PB1, and M1 that are critical for the anti-IFN functions. SA#2 is to utilize WT and mutant viral proteins containing IFN-sensitive mutations to determine the cellular proteins interacting with the anti-IFN motifs. In SA#3 is to determine the functional interaction and its biological impact in vitro and in vivo. The completion of proposed project will reveal mechanism for the anti-IFN strategy used by influenza virus, advance our understanding of influenza pathogenesis, and provide valuable targets for more effective influenza vaccine and anti-viral therapy.

项目摘要在美国，流感病毒导致5-20％的人口中的5-20％的住院治疗。流感病毒的抗原漂移和转移对流感疫苗接种计划构成了严重的挑战。虽然IFN 反应被称为控制流感病毒复制的第一个重要的宿主捍卫机制不同的步骤，流感病毒已经获得了许多抑制/对抗IFN功能的策略允许在主机中复制。众所周知，流感病毒非结构蛋白1拮抗IFN 通过抑制IFN生产来发挥作用。但是，关于流感病毒如何抵消的知之甚少 IFN刺激的基因（ISG）的功能，其中许多具有抗病毒功能。我们最近开发了一个定量和高通量基因组学系统已使新型IFN敏感的鉴定跨流感基因组的多个病毒片段的突变。该项目的目的是确定流感病毒通过研究最近确定的新型IFN敏感的抗病毒机制拮抗宿主的抗病毒机制在体外和体内使用全面的基因组和蛋白质组学平台的突变。研究计划侧重于有关流感病毒蛋白PA，PB1和M1的三个基序。每个主题，由多个代表位于小表面积的突变可能靶向细胞蛋白或IFNR信号的步骤路径。此外，我们的初步数据表明M1基序与IFN产生相互作用的突变途径，而PA和PB1上的图案可能会抵消IFN产生的下游ISG。该假设是流感PA，PB1和M1的这三个抗IFN基序与细胞蛋白相互作用以拮抗IFN 功能。一系列补充方法，包括高密度诱变，mRNA显示深度测序，ISG cDNA库筛选，CRISPR/CAS9基因编辑，人类肺原发性培养系统和基因敲除小鼠是在三个独立的，而逻辑上连接的特定目的（SAS）进行测试的我们的科学假设。 SA＃1是在病毒蛋白PA，PB1和M1上定义基序，这对于抗IFN功能。 SA＃2是利用含有IFN敏感突变的WT和突变病毒蛋白确定与抗IFN基序相互作用的细胞蛋白。在SA＃3中是确定功能相互作用及其在体外和体内的生物学影响。拟议项目的完成将揭示流感病毒使用的抗IFN策略机制，促进我们对流感的理解发病机理，并为更有效的流感疫苗和抗病毒疗法提供了宝贵的靶标。