Influenza A nonstructural gene, NS1, promotes infection and viral maturation by suppressing the host immune system. Our goal is to explore cellular targets of NS1 and determine how they are affected during infection. In addition, since current treatments to inhibit influenza infection or reduce symptoms are rather limited, we also plan to explore alternative ways to suppress infection by creating dominant negative peptides of NS1. This is a potentially powerful method to combat infection and could, in principal, be applicable to all viruses. A Drosophila stock carrying a genomic P-element insertion of UAS-NS1 was created in which expression can be locally activated with tissue specific GAL4 drivers. Phenotypes obtained from wings expressing NS1 suggest that NS1 enhances Hedgehog (Hh) signaling, a highly conserved pathway required for development of all multicellular organisms and the cause of cancer when misregulated. One goal of this application is to identify what aspect of Hh signaling is altered by NS1 and what target genes are affected. This will be explored by performing genetic epistasis tests with Hh signaling components and targets. Although, Drosophila is a highly conserved organism, it is necessary to confirm these findings in mammalian systems to ensure this is a genuine interaction. Another goal is to create potential influenza treatments by generating dominant negative NS1 alleles by treating flies carrying the NS1 transgene with Ethyl Methanesulfonate (EMS) or by expressing the P-element Transposase. Alleles will be screened for their ability to suppress the viral phenotype when co-expressed with wild type NS1. Only promising dominant negative peptides that do not cause phenotypes on their own will be tested in mammalian systems to evaluate their potential as influenza treatments. The benefit of using Drosophila for these initial screens is that it is an inexpensive, complex, multicellular organism that can be analyzed for cell autonomous and cell non-autonomous defects. It is also relatively high-throughput and highly analytical since many individuals can be examined.
Influenza A has been the cause of major pandemics including the recent deadly outbreak of the H5N1 highly pathogenic avian strain. This study has significant benefits to the public health in that it will provide an invaluable understanding of how influenza seizes the host via manipulation of a highly crucial signaling pathway. In addition, there is urgency for production of effective treatments. The dominant negative peptide inhibitors generated from this research may provide protection where available vaccines fall short and it is possible that they may even suppress infection of many different related influenza strains concomitantly.
