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Project PicoStructure

PicoStructure – Structural studies of human picornaviruses directed towards development of anti-viral compounds

ABSTRACT

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.

Figure 1.Overview of picornavirus replication cycle. Virus is endocytosed after attachment to receptors (1). There are two possible mechanisms for genome release. VP4 (yellow) and N-termini of VP1 (green) released from particles either induce endosome disruption (2a) or from a transmembrane pore for genome (red ribbon) release (2b). Genome is translated into polyprotein (blue ribbon) (3), which is cleaved into subunits (4). Viral proteins induce formation of “virus replication factories” from internal cellular membranes (5). RNA dependent RNA polymerases 3D^pol are shown as orange circles. Virions form either by encapsidation of genomes into pre-formed capsids (6a) or by assembly of particles around genomes (6b). Progeny virions are released from cells (7).

PROJECT AIMS

1. Determine virion structures of representative human viruses from genera Parechovirus, Kobusvirus, Cosavirus and species HRV-C. Study structures of virions in complexes with capsid-binding inhibitors.
2. Determine the mechanisms of picornavirus genome release from virion and delivery across biological membrane.
3. Structural analysis of picornavirus assembly and replication.