title: The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.
abstract: 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 Km.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Tanner
authors.forename: Rory M
authors.initials: RM
authors.lastname: Watt
authors.forename: Yu-Bo
authors.initials: YB
authors.lastname: Chai
authors.forename: Lin-Yu
authors.initials: LY
authors.lastname: Lu
authors.forename: Marie C
authors.initials: MC
authors.lastname: Lin
authors.forename: J S Malik
authors.initials: JS
authors.lastname: Peiris
authors.forename: Leo L M
authors.initials: LL
authors.lastname: Poon
authors.forename: Hsiang-Fu
authors.initials: HF
authors.lastname: Kung
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Huang
mesh_terms: D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D004265:DNA Helicases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D043583:Nucleoside-Triphosphatase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014709:Vero Cells
chemical_list: D004279:DNA, Viral; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases; D020365:RNA Helicases