Viruses infect their respective hosts efficiently through a regulated process of recognizing specific receptors and subsequently transferring genomic material across cell membrane barriers. To understand the underlying mechanisms that control virus infection, it is important to characterize viral events in a biologically relevat manner. I have been able to study Sf6 infection in the context of its host, Shigella flexneri, through classic phage analyses and have identified two receptors important for Sf6 infection (S. flexneri outer membrane proteins A & C, Omps A & C). OmpA is the preferred receptor but Sf6 can utilize OmpC as an alternate route for infection. I plan to study the interactions between Sf6 and S. flexneri to determine regions in both the phage proteins and the Omps that are critical for proper infection. I also plan to extend our understanding of Sf6 infection dynamics since this phage mimics infection in vitro by binding and delivering its genome into host-derived Outer Membrane Vesicles (OMVs). This provides an ideal system for using cryo-electron tomography to visualize intermediates that arise during Sf6 infection, and variant receptors as the OMVs are considerably thinner (~100 nm thick) compared to whole Shigella cells (~2000 nm thick), which are currently outside the limits of effective cryo-tomography. In vitro genome ejections are also possible using purified receptors. This makes Sf6 one of a few model systems to correlate structural transitions that arise during host cell recognition and resulting genomic transfer, and s currently the only model system for Adenovirus with alternate receptors known. We developed a time-lapse fluorescence assay that monitors real time genome ejection in vivo at the single particle level. This, combined with decades of groundwork on bacteriophage genetic manipulation and biochemical/biophysical characterization, allows us to study the process of Sf6 infection from several different angles and will provide insight into the generalized mechanisms by which all viruses recognize, and infect hosts. Sf6, as well as many other phages and eukaryotic viruses, has several so-called ejection proteins that are passed from the virion into the host during infection. Ejection proteins of Sf6 are of particular interest since they interact intimately with the host, which is a human pathogen. The location of ejection proteins pre- or post-infection has yet to be determined for any phage, but it is likely that they play a role in protecting the genome as it is injected into the host. Therefore, we predict the ejection proteins to be released in a sequential manner, and to affect the rate and efficiency of genomic transfer. We will test this hypothesis through biophysical approaches.
Virus infection is a world-wide epidemic resulting in numerous human diseases, and vast agricultural impacts. To understand how viruses commandeer hosts, it is necessary to understand the steps of host recognition and genome injection. This proposal will contribute significantly towards our understanding of these steps in a model system (where bacteriophage Sf6 infects Shigella flexneri) through coordination of biochemistry, genetics and structural studies.
