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Journal of Virology, August 2006, p. 7894-7901, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00467-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Crystal Structure of Nonstructural Protein 10 from the Severe Acute Respiratory Syndrome Coronavirus Reveals a Novel Fold with Two Zinc-Binding Motifs{dagger}

Jeremiah S. Joseph,1,2,{ddagger} Kumar Singh Saikatendu,1,2,{ddagger} Vanitha Subramanian,1,2 Benjamin W. Neuman,3 Alexei Brooun,1 Mark Griffith,2 Kin Moy,2 Maneesh K. Yadav,1 Jeffrey Velasquez,2 Michael J. Buchmeier,3 Raymond C. Stevens,2 and Peter Kuhn1*

Department of Cell Biology,1 Department of Molecular Biology,2 Department of Neuropharmacology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 920373

Received 6 March 2006/ Accepted 23 May 2006

The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 "nonstructural" proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 Å as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular ß-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the "gag-knuckle fold group" of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

* Corresponding author. Mailing address: 10550 N. Torrey Pines Rd., CB265, The Scripps Research Institute, La Jolla, CA 92037. Phone: (858) 784-9114. Fax: (858) 784-8996. E-mail: pkuhn{at}scripps.edu.

{dagger} TSRI manuscript 17990-CB.

{ddagger} These authors contributed equally to this work.

Journal of Virology, August 2006, p. 7894-7901, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00467-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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