The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor

Abstract

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in Escherichia coli has endoribonuclease activity that preferentially cleaved 5 of uridylates of RNAs. Blocking either the 5 or 3 terminus did not affect cleavage. Double-and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn 2؉ at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg 2؉ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn 2؉ needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…Because of the large size of CoV genomes, which until recently has limited the use of reverse genetics approaches, the activities and functions of the various enzymes in the CoV life cycle are only slowly beginning to emerge. Recently, a CoV endoribonuclease activity has been identified and shown to have a critical role in CoV replication and transcription (14,15). Here, we report that CoVs encode a second ribonucleolytic activity.…”

Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation

“…These precursors undergo proteolytic processing via two virally encoded proteinases to generate 16 mature proteins, nsp1 through nsp16. Probably most of the gene 1 proteins are important for viral RNA synthesis (5,(7)(8)(9)(10)(11). Some gene 1 proteins are predicted to have biological functions related to RNA synthesis (11), and some have demonstrated functions that appear to be involved in RNA synthesis (7,8,10,12), whereas some gene 1 proteins may have functions other than viral RNA synthesis (13)(14)(15)(16).…”

Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant fromHuman coronavirus 229E

“…Nsp15, the only endoribonuclease residing in the genome, is considered to be a major 'genetic marker' of nidoviruses as it is not found in other RNA viruses (Ivanov et al, 2004). Nsp15 has critical functions in viral replication and transcription, although how nsp15 acts in the viral cycle remains unclear (Bhardwaj et al, 2004;Ivanov et al, 2004). A mutation in nsp15 of HCoV-229E has been reported to prevent viral RNA accumulation, suggesting that nsp15 is (Ivanov et al, 2004).…”

“…Nonstructural protein 15 (nsp15), a gene product of ORF 1b, has been identified as an endoribonuclease (Bhardwaj et al, 2004). Nsp15, the only endoribonuclease residing in the genome, is considered to be a major 'genetic marker' of nidoviruses as it is not found in other RNA viruses (Ivanov et al, 2004).…”

“…In addition, the structure of the monomeric nsp15 in SARS-CoV was determined through an N-terminal domain truncation (PDB entry 2ozk; Joseph et al, 2007). Hexamers have been considered to be the fully active form, while monomers lose the enzymatic activity owing to a lack of RNAbinding ability (Bhardwaj et al, 2004;Joseph et al, 2007). In previous investigations on nsp15s, even though several sitedirected mutations were made, trimers only existed in the transition state between hexamers and monomers.…”