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Journal of Virology, November 2004, p. 12218-12224, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12218-12224.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Kanchan Bhardwaj, Linda Guarino, and C. Cheng Kao*

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas

Received 23 April 2004/ Accepted 9 July 2004

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. Mn2+ at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg2+ and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn2+ 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.

* Corresponding author. Mailing address: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128. Phone: (979) 458-2235. Fax: (979) 845-9274. E-mail: ckao{at}tamu.edu.

Journal of Virology, November 2004, p. 12218-12224, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12218-12224.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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