Mechanism for anti-interferon functions of influenza virus

项目摘要 在美国，甲型流感病毒在5-20%的人口中引起疾病，每年有超过200，000人住院。 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IFN 应答被认为是控制流感病毒复制的第一个重要的宿主防御机制， 不同的步骤，流感病毒已经获得了许多策略来有效地抑制/拮抗IFN功能 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IFN 通过抑制IFN的产生发挥作用。然而，对流感病毒如何抵消 干扰素刺激基因（ISG）的功能，其中许多具有抗病毒功能。我们最近开发的一种 定量和高通量基因组学系统已能够识别新型干扰素敏感性 流感病毒基因组中多个病毒片段的突变。这个项目的目标是确定如何 流感病毒拮抗宿主抗病毒机制研究新发现的干扰素敏感 使用全面的基因组和蛋白质组学平台在体外和体内研究突变。研究计划 重点关注流感病毒蛋白PA、PB 1和M1上的三个基序。每个主题，由多个 突变位于一起在一个小的表面积，可能针对细胞蛋白质或步骤的IFNR信号 通路此外，我们的初步数据表明M1基序上的突变与IFN的产生相互作用 PA和PB 1上的基序可能抵消IFN产生下游的ISG。这个假设是 流感病毒PA、PB 1和M1的这三种抗IFN基序与细胞蛋白相互作用以拮抗IFN 功能协调发展的一系列互补的方法，包括高密度诱变、mRNA深度展示 测序、ISG cDNA文库筛选、CRISPR/Cas9基因编辑、人肺原代培养系统，以及 基因敲除小鼠提出了三个独立的，而逻辑连接的特定目的（SA）进行测试 我们的科学假设SA#1是定义病毒蛋白PA、PB 1和M1上的基序，这些基序对病毒的免疫至关重要。 抗干扰素功能。SA#2是利用含有IFN敏感性突变的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，确定函数 相互作用及其在体外和体内的生物学影响。该项目的完成将揭示 针对流感病毒所采用的抗IFN策略的机制，推进我们对流感的认识 致病机理的研究，为更有效的流感疫苗和抗病毒治疗提供了有价值的靶标。

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.