NMR studies on the molecular regulation of proteinase 3c from Picorna viruses

The chymotrypsin-like cysteine ​​proteinases 3C play a central role in picornavirus gene expression and replication and can therefore serve as a target for antiviral therapy. Their bifunctionality as proteases and RNA-binding proteins suggests that they control the opposing and competitive processes of viral protein and RNA synthesis in the viral life cycle and possibly act as molecular switches in the various steps of this cycle. Atomic details of the binding of picornaviral RNA to proteinase 3C are currently unknown. In particular, the mutual influence of RNA binding and binding of ligands in the active site remains unexplored. NMR experiments will be used to investigate the binding of viral RNA and active site ligands to proteinase 3C. First, the RNA binding site will be localized at atomic resolution, making use of known crystal structure analysis data. Based on this, possible allosteric interactions between RNA and ligand binding will be investigated. Further NMR experiments will be conducted to determine the bioactive conformations and binding epitopes of the ligands. Complementing the NMR studies, thermodynamic and kinetic parameters of the binding reactions will be determined using surface plasmon resonance. Since nucleic acid-binding proteases are also active in other viruses, such as hepatitis C or adenovirus, the findings will be of general virological interest.