X-ray crystal structure, proteolytic dependent activation, and functional analysis of the poliovirus RNA-dependent RNA polymerase

Abstract

Plus-strand RNA viruses are responsible for a large number of human and animal diseases including poliomyelitis, yellow fever, hepatitis C, foot-and-mouth disease, and dengue fever. Upon infection, the single plus-strand RNA molecule contained within the viral particle is translated by cellular ribosomes of the host into a single large polyprotein that is then cleaved by viral proteases into 11 distinct proteins and several functional precursors. The last enzyme in this polyprotein is a RNA-dependent RNA polymerase that is required for replication of the viral genome. Using a novel method of intentionally disrupting a known and persistent crystal packing interaction, we have solved the crystal structure of the complete poliovirus polymerase and complexes containing all four ribonucleotides and Mn2+. The structures show the conformation of the previously unresolved "fingers" domain and reveal that the very N-terminus of the protein is buried in a pocket on the surface of the protein. This proteolytic dependent burial of the N-terminus forms important hydrogen bonds that are required for the proper positioning of a key residue in the active site, Asp238, which in turn forms a required hydrogen bond with the 2' OH group of the incoming NTP. Additionally, we provide data showing that interdomain interactions responsible for the "enclosed" active site of this enzyme are important for thermal stability. These data suggest that poliovirus 3Dpol and other viral RNA-dependent RNA polymerases are unusually rigid, and we thus propose an exposed portion of the protein (pinky finger; residues 96-149) may play a significant role in the nucleotide polymerization activity of the poliovirus polymerase.