Structural basis for proteolysis-dependent activation of the poliovirus RNA-dependent RNA polymerase

The active RNA-dependent RNA polymerase of poliovirus, 3D(pol), is generated by cleavage of the 3CD(pro) precursor protein, a protease that has no polymerase activity despite containing the entire polymerase domain. By intentionally disrupting a known and persistent crystal packing interaction, we have crystallized the poliovirus polymerase in a new space group and solved the complete structure of the protein at 2.0 Å resolution. It shows that the N-terminus of fully processed 3D(pol) is buried in a surface pocket where it makes hydrogen bonds that act to position Asp238 in the active site. Asp238 is an essential residue that selects for the 2′ OH group of substrate rNTPs, as shown by a 2.35 Å structure of a 3D(pol)–GTP complex. Mutational, biochemical, and structural data further demonstrate that 3D(pol) activity is exquisitely sensitive to mutations at the N-terminus. This sensitivity is the result of allosteric effects where the structure around the buried N-terminus directly affects the positioning of Asp238 in the active site

306 - Colleen Lanza Watkins

The picornavirus family of viruses includes poliovirus, the causative agent of paralytic polio and coxsackievirus, which is responsible for viral-heart-disease. Picornaviruses contain a single-stranded positive-sense RNA genome replicated by 3Dpol, an RNA-dependent RNA polymerase (RdRP). Crystal structures of 3Dpol from multiple picornaviruses have shown a conserved polymerase fold analogous to a “right hand” composed of fingers, palm and thumb domains. These crystal structures also identified unique regions in the fingers domain whose function in 3Dpol were unknown. Through biochemical kinetic analysis we have now determined the purpose of these regions, and their effects on the catalytic cycle of 3Dpol.Great Minds in Research - Honorable Mention

Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability ▿

The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex