Mechanism for anti-interferon functions of influenza virus

项目摘要 甲型流感病毒导致5%-20%的人口患病，美国每年有超过20万人住院治疗。 流感病毒的抗原漂移和转移对流感疫苗接种计划提出了严峻的挑战。虽然干扰素 应答被称为控制流感病毒复制的第一个重要宿主防御机制 在不同的阶段，流感病毒已经获得了许多有效抑制/拮抗干扰素功能的策略 允许其在主机中复制。众所周知，流感病毒非结构蛋白1拮抗干扰素 通过抑制干扰素的产生发挥作用。然而，人们对流感病毒如何起作用知之甚少。 干扰素刺激基因(ISGs)的功能，其中许多具有抗病毒功能。我们最近开发了一种 定量和高通量基因组学系统使新的干扰素敏感性的鉴定成为可能 流感基因组中多个病毒片段的突变。该项目的目标是确定如何 流感病毒通过研究新近发现的干扰素敏感新基因拮抗宿主抗病毒机制 在体外和体内使用全面的基因组和蛋白质组平台进行突变。研究计划 聚焦于流感病毒蛋白PA、PB1和M1的三个基序。每个主题，由多个 位于一小块表面积内的突变可能针对细胞蛋白或IFNR信号的步骤 路径。此外，我们的初步数据表明，M1基序上的突变与干扰素的产生相互作用 途径，而PA和PB1上的基序可能抵消干扰素下游的ISGs。假设是 流感病毒PA、PB1和M1的这三个抗干扰素基序与细胞蛋白相互作用而拮抗干扰素 功能。一系列互补的方法，包括高密度突变、mRNA展示深度 测序，ISG文库筛选，CRISPR/Cas9基因编辑，人肺原代培养系统，以及 基因敲除小鼠被提出在三个独立但逻辑相连的特定目标(SA)中进行测试 我们的科学假设。SA#1是定义病毒蛋白PA、PB1和M1上的基序，这些基序对 抗干扰素功能。SA#2是利用WT和含有干扰素敏感突变的突变病毒蛋白来 确定与抗干扰素基序相互作用的细胞蛋白。SA#3中的目的是确定功能 体内外相互作用及其生物学影响。拟议项目的完成将揭示 流感病毒抗干扰素策略的作用机制，加深对流感的认识 并为更有效的流感疫苗和抗病毒治疗提供有价值的靶点。

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.