The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.
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.
Tanner
Julian A
JA
Department of Biochemistry, Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China.
Watt
Rory M
RM
Chai
Yu-Bo
YB
Lu
Lin-Yu
LY
Lin
Marie C
MC
Peiris
J S Malik
JS
Poon
Leo L M
LL
Kung
Hsiang-Fu
HF
Huang
Jian-Dong
JD
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
D004279:DNA, Viral; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases; D020365:RNA Helicases