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 crosslinking, 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.The Nidoviruses contain three families of viruses, including the Coronaviridae that cause numerous diseases in humans (1). Severe acute respiratory syndrome coronavirus (SARS-CoV) 3 is a member of the Coronavirus genus (2, 3). It originated from animals but spread to humans, causing severe respiratory distress with a fatality rate of ϳ10% (as shown by the World Health Organization, www.who.int/csr/ sars/country/en/country2003_08_15.pdf). In addition to their medical importance, coronaviruses are of interest for their large ϳ30-kb positive-strand genome and novel mechanisms that have evolved to replicate and transcribe this large RNA (5, 6). In keeping with the novel strategies used, coronaviruses encode several unusual RNA-processing enzymes, including an RNA endoribonuclease, an RNA methyltransferase, a second RNA-dependent RNA polymerase that generates primers for coronavirus replication (7), and a mechanism to decrease replication errors (8).Coronavirus subgenomic RNAs are particularly interesting in that they all have the same 5Ј leader sequence derived from the 5Ј end of the genomic RNA. This organization requires recombination as part of transcription. Various mechanisms have been proposed for subgenomic RNA production, but a discontinuous transcription mechanism is increasingly favored (5). This model proposes that transcription regulatory sequences in the minus-strand RNA direct translocation of the ternary complex to the 5Ј leader sequence, where transcription resumes. The minus-strand RNAs serves as the template for subgenomic RNA transcription. Ribonucleases that process the RNA intermediates for transcription have been proposed (9 -11), but the mechanism for the process is still not completely understood.Nsp15 (nonstructural protein 15) was predicted to be an RNA endoribonuclease as part of a bioinformatics analysis of the SARSCoV genome (7). S...

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“…After reversal of the cross-links with heat, the peptides are subjected to mass spectrometry and the results are compared to those for the peptides generated from a theoretical digest of the protein. For proteins with known structures, the peptides can be immediately mapped back to the three-dimensional (3D) model of the protein (3,21,30).…”

The Coronavirus Endoribonuclease Nsp15 Interacts with Retinoblastoma Tumor Suppressor Protein

“…Furthermore, cleavage occurs through the formation of a 2=-3= cyclic phosphodiester product in a mechanism identical to that of RNase A (4). Crystal structures of the SARS-CoV and the mouse hepatitis virus (MHV) Nsp15 have been reported, and both proteins form hexamers in solution (3,36,42).…”

“…Furthermore, cleavage occurs through the formation of a 2=-3= cyclic phosphodiester product in a mechanism identical to that of RNase A (4). Crystal structures of the SARS-CoV and the mouse hepatitis virus (MHV) Nsp15 have been reported, and both proteins form hexamers in solution (3,36,42).Mutations in the active site of Nsp15 that apparently abolish endoribonuclease activity in vitro reduce viral infectivity by up to 2 logs (20, 35). However, some mutations outside of the active site are reported to have a larger effect on virus viability, suggesting that Nsp15 has role(s) in coronavirus infection apart from its function as an endoribonuclease (18,20).…”

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