Highly virulent H5N1 strains of influenza A virus are currently circulating in a number of avian species inAsia, Europe, and Africa. A devastating pandemic is possible if these strains acquire the ability for efficienthuman-to-human spread. A recent study found that the viral NS1A protein is associated with the virulence ofH5N1 viruses. It was also found that avian influenza NS1A proteins, as well as NS1A proteins from recenthuman H5N1 infections, contain a domain at their carboxyl termini termed the PDZ-ligand domain, and thisdomain associates in vitro with cellular PDZ proteins. PDZ proteins are a large class of proteins that aretypically involved in cell-cell contract. It is therefore possible, if not likely, that the association of these NS1Aproteins with key cellular PDZ proteins is an aspect of virulence in vivo. To develop an assay fortherapeutics 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 theNS1A PDZ-ligand domain and its cellular PDZ protein targets. This screen will be based upon a recentlydescribed 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 WCREmission.
Specific Aim 2 : To identify cellular PDZ proteins that associate in vivo with the PDZ-ligand domain of theavian influenza virus NS1A protein. The identification of these cellular proteins will be accomplished by coimmunoprecipitationwith 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 perturbprotein-protein interactions. These publications have therefore established the feasibility of drugs thatFunction by interfering with protein-protein interactions. Completion of the work proposed here will provide anassay for small molecules that block the interaction of the NS1A PDZ-ligand domain with its targets, thuslaying the foundation for development of novel therapeutics for H5N1 infections.
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