Biochemical and structural studies exploring the mechanism of poliovirus cell entry

Poliovirus, a member of the picornavirus family, was utilized as a model system for studying non-enveloped viruses. One of the great enigmas surrounding non-enveloped virus infection is how the virus particle or its genome crosses the membrane. To answer this question, I solved the structure of the 135S intermediate to 11A resolution and developed a membrane system to study both the biochemistry and structural biology of the early stages in poliovirus entry. The higher resolution 135S structure compared with a protease-cleaved 135S reveals the location of a peptide that becomes externalized during entry and inserts into membranes. Docking and refinement of native viral protein x-ray coordinates into the 135S map described the transition from native to 135S specifically focusing on the egress pathway of this peptide. A membrane model system was used to study earlier stages of entry. Poliovirus is associated with the membrane through its receptor, Pvr, which has been tethered to NTA-liposomes. This model system was linked to events during infection by the observation that virus bound to receptor-decorated liposomes could form biologically relevant entry intermediates. Structural studies utilizing NTA-liposomes offer the first look into the early stages of poliovirus entry within the context of a lipid environment. The structure of the virus-receptor-liposome complex, solved by cryo-electron microscopy, reveals that five receptors are required for attachment and the viral 5-fold axis, consisting of VP1, is directed towards the membrane. Although the 5-fold axis of picornaviruses has been thought to be important in pore formation and RNA translocation, this structure is the first experimental evidence confirming this hypothesis. These findings not only describe the initial entry intermediate for poliovirus, but have broader implications for related picornavirus entry mechanisms. The mixed symmetry of the virus-receptor-liposome complex posed computational challenges that were overcome by the development of new methodologies in single-particle image processing. The NTA-liposome system combined with new computational techniques developed to solve these types of membrane bound structures are powerful tools that can be applied not only to studying the later stages of poliovirus entry, but present a novel technique for studying membrane bound complexes as a whole.