Influenza A virus interactions with host cells: From cell tropism to nuclear targeting

This thesis examines host-pathogen interaction between influenza virus and target cells. Two aspects of the virus life cycle were examined; first the initial interaction of influenza virus with respiratory epithelial cells, and second how cellular localization of the influenza NS1 protein affects protein function.;To examine the virus recognition of epithelial cells, primary differentiated epithelial cultures offer a tissue relevant system with multiple cell types similar to in vivo tissue. As a part of this project, primary differentiated respiratory epithelial cell culture systems were developed from rhesus macaque nasal and tracheal tissue. Non human primates are an important animal model in influenza research as well as other infectious and non infectious diseases, but no primary respiratory epithelial culture system existed. The cultures were optimized and characterized and were able to robustly support influenza infection. The cultures supported viruses with human type influenza receptor recognition better than viruses with avian type.;The study of cellular localization of the influenza NS1 protein involved using reverse genetics to introduce mutations into the virus and plasmid expression of the NS1 protein. The highly conserved N terminal NLS sequence between amino acids 35 and 41 was examined. Even though mutating individual NS1 has been shown to interfere with recognition by nuclear import pathways (Melen et al. 2007), NS1 proteins with NLS mutations were able to enter the nucleus. When spontaneous mutations to D39 (D39X) arose appearing to compensate for loss of function in the NS1 R35A virus rescue attempts, it was hypothesized that these mutations were compensating for loss in NS1 protein dimerization. Protein dimerization was investigated using a plasmid based reporter system that produced functional florescent YFP upon NS1 dimerization, and it was demonstrated that proteins expressing both R35A and D39X mutations dimerized better than the R35A mutation alone, though not to the same extent as wild type NS1.;Despite available vaccines and antiviral drugs, influenza remains a common infectious disease. This thesis expands the understanding of how the virus and host cell interact both at the initial steps of cell and receptor tropism as well as the intermolecular interaction important to function of the influenza interferon antagonist, the NS1 protein.