"Many picornaviruses are human pathogens that cause diseases varying in symptoms from common cold to life-threatening encephalitis. Currently there are no anti-picornavirus drugs approved for human use. We propose to study molecular structures of picornaviruses and their life cycle intermediates in order to identify new targets for anti-viral inhibitors and to lay the foundations for structure-based development of drugs against previously structurally uncharacterized picornaviruses.We will use X-ray crystallography to determine virion structures of representative viruses from Parechovirus, Kobuvirus, Cardiovirus, and Cosavirus genera and Human Rhinovirus-C species. We will use cryo-electron microscopy to study picornavirus replication complexes in order to explain the mechanism of copy-choice recombination of picornavirus RNA genomes that leads to creation of new picornavirus species. We will determine whether picornavirus virions assemble from capsid protein protomers around the condensed genome or if the genome is packaged into a pre-formed empty capsid. Furthermore, we will investigate how picornaviruses initiate infection by analyzing genome release from virions and its translocation across lipid membrane.A major innovation in our approach will be the use of focused ion beam micromachining for sample preparation that will allow us to study macromolecular complexes within infected mammalian cells by cryo-electron tomography. Our analysis of virion structure, cell entry, genome replication, and particle assembly will identify molecular details and mechanism of function of critical picornavirus life-cycle intermediates."