Dataset Information

Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15.

ABSTRACT: The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.

SUBMITTER: Bhardwaj K

PROVIDER: S-EPMC8740563 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Nonstructural protein 15 (Nsp15) encoded by coronavirus (CoV) is a nidoviral uridylate-specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural information on this crucial protein from the Middle East respiratory syndrome CoV (MERS-CoV), which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we determined the crystal structure of MERS-CoV Nsp15 at a 2.7-Å resolution and performed the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with that of other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A, and N157A, decrease thermostability, decrease affinity of binding with RNA, and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 was found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15 in vitro Taking the findings together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency, or the possible association with other nonstructural proteins.IMPORTANCE The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and the Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.

Dataset Information

Structural and functional analyses of the severe acute respiratory syndrome coronavirus endoribonuclease Nsp15.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets