"Viral infections cause problems worldwide and result in a multitude of human diseases ranging in severity from influenza to HIV. Most viruses infect their respective host cells by attaching to a receptor, ejecting their genomes, and replicating via the host cell machinery. The step of attachment and entry requires precise docking to a designated location on the surface of the cell followed by conformational changes that result in viral genome transfer into the cell. However, the molecular mechanisms that drive receptor binding have not been elucidated for the vast majority of viruses. Because viral protein structures and mechanisms are conserved across families of viruses, Sf6 and its host Shigella flexneri, may be used as a model system to provide insight into understanding viral attachment to host cells. Sf6 uses lipopolysaccharide (LPS) as a primary (1o ) receptor for an initial reversible, interaction, and it requires a secondary (2o ) irreversible receptor to commit to infection. Both outer membrane proteins A and C (OmpA and OmpC) may serve as secondary receptors for Sf6, although OmpA is slightly preferred. Here, we investigate how bacteriophage Sf6 utilizes OmpA and OmpC for infection. First, we identified that the surface loops of OmpA are important for Sf6 infection. Using a combination of in vivo and in vitro approaches including, but not limited to, phage plaque assays, site-directed mutagenesis, circular dichroism spectroscopy, and in vitro genome ejection assays we characterized which residues in the surface loops of OmpA are responsible for productive Sf6 infection. We showed that individual amino acid substitutions have a range of effects implicating some locations in the loops as more important than others for infection. Next, we used BioLayer Interferometry (BLI), an optical biosensing technique, to determine binding affinities of Sf6 to OmpA and single substitution variants. We immobilized whole virions and determined the kinetic parameters of Sf6 to various OmpAs to be fast-on and slow-off. The binding affinity of Sf6 to S. flexneri OmpA is in the low nM range. We also show that Sf6 binds to five variant OmpAs and the resultant kinetic parameters vary only slightly. These kinetic data suggest that Sf6:Omp receptor recognition is not solely based on kinetics, but potentially on the ability of the Omp to induce the correct conformational changes in the virion which result in translocation of the DNA. Finally, we purified OmpC and using a limited proteolysis approach, we obtained trypsin resistant and functional trimeric OmpC. The resultant OmpC, in combination with LPS, causes Sf6 genome ejection in vitro, but at a lower efficiency and rate than with OmpA. Taken together, the data presented in this dissertation shed light on how Sf6 interacts with its secondary receptors, OmpA and OmpC, an important aspect of host recognition, and provide new insights into Podoviridae attachment."--Pages ii-iii.
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