The NS1 protein is the only non-structural protein coded for by the influenza virus. Through the use of reverse genetics, a viable transfectant influenza A virus (de1NS1), which lacks the NS1 gene, has been generated. When the growth properties of de1NS1 and the isogenic wild- type influenza viruses were compared, it was observed the de1NS1 was attenuated for growth in hosts which were capable of eliciting an interferon response to viral infection. However, de1NS1 virus grew to higher titers and was much more virulent in systems that were defective or immature in their interferon response systems. The results with de1NS1 virus suggest that the NS1 protein of influenza A viruses is a virulence factors which plays a crucial role as an antagonist of the type I interferon-mediated antiviral responses of the host. We will construct and recovery recombinant influenza A viruses in order to determine which domains on the NS1 protein are required for and/or contribute to interferon antagonism. Several domains were previously mapped on the NS1 protein and include (1) an RNA binding domain, (2) an effector function, (3) a dimerization domain and (4) two nuclear localization signals We will take advantage of the novel plasmid-only rescue system to recover viruses in which the NS1 ORF is site- specifically altered or specific domains are ablated. Rescued recombinant influenza viruses will then be characterized for their ability to grow in interferon-competent hosts (i.e., 10-day-old embryonated eggs and in intranasally inoculated mice). Since the NS1 protein can stimulate the expression of a reporter luciferase construct in transfected cells, mutant NS1 proteins will also be characterized for their ability to stimulate the expression the luciferase gene. We will also construct a de1NS1-like influenza A/Texas/36/91 virus for study in humans. These latter studies will be carried out in conjunction with Dr. Treanor's group. In a second line of experiments we will determine whether the influenza B virus NS1 protein is also an interferon antagonist. To do this we will establish a plasmid-only rescue system for influenza B viruses. Altered influenza B virus NS1 genes will be recovered into virus, and examine for their ability to grow into the embryonated egg and mouse systems.
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