Molecular components specifying mucosal cell sensitivity to influenza infection

描述（申请人提供）：通过更广泛地理解被破坏以支持病毒感染的细胞自主分子组分以及被动员以防御病毒感染的细胞自主分子组分，将极大地促进转向或改善流感感染的新策略的开发。全基因组siRNA文库的出现，加上单孔/单基因高通量筛选策略，提供了产生实验平台的机会，以获得支持关键生物系统的经验证的基因靶点的无偏见的全面集合。我们的方法是应用基于全基因组RNAi的体细胞遗传学，对人支气管上皮细胞中支持1）流感生命周期和2）抗病毒监测和反应系统的基因产物进行广泛和无偏见的鉴定。在特定目标1中，将利用全基因组siRNA筛选来鉴定在暴露于甲型流感病毒/WS/33时使支气管上皮细胞死亡转向（抵抗基因座）或促进（致敏基因座）的所有基因缺失。在具体目标2中，将采用重点分层实验策略将验证的命中解析为代表关键生物过程的功能互补组。控制流感病毒复制和宿主先天免疫途径激活的偏转的关键生物压力点的所得分子注释将用于帮助1）产生对宿主/病毒关系的新的机制见解，2）拓宽支持病毒复制的宿主系统中的可靶向生物压力点的知识，和3）有助于鉴定具有药物样性质的新型生物活性化合物的分子靶标。流感病毒必须劫持存在于人类粘膜细胞内的蛋白质，以便在受感染的个体中繁殖。详细了解支持病毒复制所需的粘膜细胞蛋白以及动员起来抵御病毒复制的粘膜细胞蛋白将有助于开发新的治疗和预防干预措施。这里描述的工作将有助于产生一个全面的知识基础的粘膜细胞蛋白，既支持和拮抗流感A感染和复制。

DESCRIPTION (provided by applicant): Development of novel strategies to deflect or ameliorate influenza infection will be greatly facilitated by a broader understanding of the cell-autonomous molecular components that are subverted to support viral infection, and those that are mobilized to defend against it. The advent of genome-wide siRNA libraries, coupled with one-well/one-gene high throughput screening strategies, provides the opportunity to generate experimental platforms to derive unbiased comprehensive collections of validated gene targets that support critical biological systems. Our approach is to apply genome-wide RNAi-based somatic cell genetics for an extensive and unbiased identification of gene products in human bronchial epithelial cells that support 1) the influenza life cycle and 2) the antiviral surveillance and response system. In Specific Aim 1, a genome-wide siRNA screen will be leveraged to identify all gene depletions that deflect (resistor loci) or promote (sensitizor loci) bronchial epithelial cell death upon exposure to influenza A/WS/33. In Specific Aim 2 a focused hierarchical experimental strategy will be employed to parse validated hits into functional complementation groups representing key biological processes. The resulting molecular annotation of critical biological pressure points controlling flu replication and deflection of host innate immune pathway activation will be used to help 1) generate novel mechanistic insights into the host/virus relationship, 2) broaden knowledge of targetable biological pressure points in host systems that support viral replication, and 3) facilitate identification of the molecular targets of novel bioactive compounds with drug-like properties. Influenza virus must hijack proteins present within the cells of the human mucosa in order to reproduce in infected individuals. A detailed understanding of the mucosal cell proteins required to support viral replication as well as those which are mobilized to defend against it will aid development of novel therapeutic and preventative interventions. The work described here will help produce a comprehensive knowledge base of the mucosal cell proteins that both support and antagonize influenza A infection and replication.