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J. Biol. Chem., Vol. 278, Issue 41, 39578-39582, October 10, 2003

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The Severe Acute Respiratory Syndrome (SARS) Coronavirus NTPase/Helicase Belongs to a Distinct Class of 5' to 3' Viral Helicases^*,

Julian A. Tanner  , Rory M. Watt  , Yu-Bo Chai , Lin-Yu Lu , Marie C. Lin ¶, J. S. Malik Peiris ||, Leo L. M. Poon ||, Hsiang-Fu Kung ¶ and Jian-Dong Huang  **

From the Department of Biochemistry, Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China, the ^||Department of Microbiology, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China, and the ^¶Institute of Molecular Biology, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15–25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT₂₄ binding over 10 times more strongly than dA₂₄ as observed by the apparent K_m.

Received for publication, July 23, 2003 , and in revised form, August 11, 2003.

The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession number NC_828870.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a Supplemental Material.

These authors contributed equally to this research.

^** To whom correspondence should be addressed. E-mail: jdhuang{at}hkucc.hku.hk.

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