Targeting the PDZ-ligand domain of avian> influenza A viruses for novel therapeut

甲型流感病毒的高毒力H5 N1毒株目前在美国的一些禽类物种中传播， 亚洲、欧洲和非洲。如果这些菌株获得了有效的 人际传播最近的一项研究发现，病毒NS 1A蛋白与病毒的毒力有关， H5 N1病毒。还发现禽流感病毒NS 1A蛋白，以及最近的NS 1A蛋白， 人H5 N1感染，在其羧基末端含有一个称为PDZ-配体结构域的结构域， 结构域在体外与细胞PDZ蛋白缔合。PDZ蛋白是一大类蛋白质， 通常与细胞间的联系有关因此，这些NS 1A的关联是可能的，如果不是可能的话， 与关键细胞PDZ蛋白结合的蛋白质是体内毒力的一个方面。开发一种检测方法， 针对这种病毒功能的治疗，提出了两个具体目标： 具体目标1：开发一种高通量筛选化学抑制剂的相互作用， NS 1A PDZ-配体结构域及其细胞PDZ蛋白靶标。此屏幕将基于最近 所描述的方法称为反向MAPPIT（哺乳动物蛋白质-蛋白质相互作用陷阱）。一旦建立， 该系统可以很容易地适用于筛选与WCRE相关的其他病原体的抑制剂 使命。 具体目的2：鉴定在体内与PDZ-配体结构域结合的细胞PDZ蛋白， 禽流感病毒NS 1A蛋白。这些细胞蛋白的鉴定将通过免疫共沉淀来完成 使用串联亲和纯化（TAP）方法与禽类NS 1蛋白进行纯化。 许多最近的出版物报道了在筛选干扰分子的小分子方面的成功。 蛋白质相互作用因此，这些出版物确定了药物的可行性， 通过干扰蛋白质-蛋白质相互作用发挥作用。完成此处建议的工作将提供 用于阻断NS 1A PDZ-配体结构域与其靶的相互作用的小分子的测定，因此 为开发H5 N1感染的新型疗法奠定了基础。

Highly virulent H5N1 strains of influenza A virus are currently circulating in a number of avian species in Asia, Europe, and Africa. A devastating pandemic is possible if these strains acquire the ability for efficient human-to-human spread. A recent study found that the viral NS1A protein is associated with the virulence of H5N1 viruses. It was also found that avian influenza NS1A proteins, as well as NS1A proteins from recent human H5N1 infections, contain a domain at their carboxyl termini termed the PDZ-ligand domain, and this domain associates in vitro with cellular PDZ proteins. PDZ proteins are a large class of proteins that are typically involved in cell-cell contract. It is therefore possible, if not likely, that the association of these NS1A proteins with key cellular PDZ proteins is an aspect of virulence in vivo. To develop an assay for therapeutics that target this viral function, two Specific Aims are proposed: Specific Aim 1: To develop a high-throughput screen for chemical inhibitors of the interaction between the NS1A PDZ-ligand domain and its cellular PDZ protein targets. This screen will be based upon a recently described method termed Reverse MAPPIT (mammalian protein-protein interaction trap). Once established, this system can be readily adapted for screens of inhibitors for other pathogens of relevance to the WCRE mission. Specific Aim 2: To identify cellular PDZ proteins that associate in vivo with the PDZ-ligand domain of the avian influenza virus NS1A protein. The identification of these cellular proteins will be accomplished by coimmunoprecipitation with the avian NS1 protein using a tandem-affinity purification (TAP) method. A number of recent publications have reported success in screens for small molecules that perturb protein-protein interactions. These publications have therefore established the feasibility of drugs that Function by interfering with protein-protein interactions. Completion of the work proposed here will provide an assay for small molecules that block the interaction of the NS1A PDZ-ligand domain with its targets, thus laying the foundation for development of novel therapeutics for H5N1 infections.