Time Resolved Structural Analysis of Macromolecules and Assemblies (360G-Wellcome-109134_Z_15_A)

The icosahedral, positive-sense RNA Foot and Mouth Disease (FMDV) remains a foe to the agricultural industry; causing vesicular disease of cloven-hoofed animals. Converse to other picornaviruses, FMDV has a significantly greater pH and temperature sensitivity. In the quest for thermostable vaccine development, many wild type and mutant structures of the seven Foot and Mouth Disease viral serotypes are now known. Despite this, the structural basis of infection, specifically at the point of RNA loss, remains elusive. We will exploit this pH sensitivity in time-controlled study of FMDV-RNA release. The primary technique will be time-resolved single particle cryo-electron microscopy, coupled with the existing arsenal of structural biology techniques. Time-resolution of single particle structures will be attempted by varying time delays between FMDV sample acidification using photo-sensitive caged-proton compound and vitrification. Time-dependent SAXs may also be employed to monitor conformational changes. Deterioration of icosahedral capsid symmetry may be captured using cryo-electron tomography. Tomography will also be applied to a model infection system, capturing FMDV binding and pH driven uncoating via a nanodisk-immobilised integrin alphavbeta6 receptor. From a methodological point of view, we hope to establish a framework that exploits the synergy between single particle, tomographic, and crystallographic methods.