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.

Fig. 1

Purification and DNA-unwinding activity of the SARS coronavirus NTPase/helicase.Panel A, purification of the SARS-CoV Hel as observed by 4–12% SDS-PAGE. Lane 1, marker, from bottom (in kilodaltons) 10, 15, 20, 25, 30, 40, 50 (strong), 60, 70, 80, 90, 100, 120, 160, 220. Lane 2, Whole cell extract from E. coli BL21 (DE3) expressing SARS-CoV Hel (5 μg). Lane 3, soluble extract from E. coli BL21 (DE3) expressing SARS-CoV Hel (10 μg). Lane 4, SARS-CoV Hel after purification by nickel affinity chromatography (1.5 μg). Lane 5, SARS-CoV Hel after purification on Red Sepharose CL-6B (0.4 μg). Panel B, schematic showing the fully double-stranded (dB) and partially double-stranded DNA substrates (d5T = 5′-oligo(dT)₂₀ tail, d3T = 3′-oligo(dT)₂₀ tail) used in the unwinding assay. An asterisk indicates the position of the ³²P radiolabel. Panel C, the SARS-CoV Hel unwinds duplex DNA with a 5′ to 3′ polarity. The positions of the intact dsDNA duplexes (dB, d5T, and d3T), as well as the released (labeled) ssDNA strand on the polyacrylamide gel, are indicated with arrows. Lanes 1–5, reactions containing 30 fmol of DNA duplex; lanes 2–5, reactions containing 6 pmol of helicase; lanes 1–4, reactions containing 2.5 mm ATP; lane 1, the d5T DNA duplex was heat-denatured. Panel D, titration of helicase concentration. Lanes 1–4, reactions containing 150 fmol, 600 fmol, 1.5 pmol, and 3 pmol of helicase, respectively, in addition to 30 fmol of d5T DNA duplex and 2.5 mm ATP.

Fig. 2

Comparative polynucleotide-induced stimulation of the ATPase activity of the SARS coronavirus NTPase/helicase. Each 24-mer polynucleotide was added to a concentration of 1 μm. The degree of stimulation was calculated as a factor, normalized to the rate of ATPase activity in the absence of polynucleotide. Experimental conditions were: 0.5 mm ATP, 5 mm MgCl₂, 50 mm Tris-HCl, pH 6.6, 50-μl reaction volume, 0.5 pmol of helicase, 10-min reaction.

Fig. 3

Effect of NTP and polynucleotide concentration on rate of NTP hydrolysis catalyzed by the SARS coronavirus NTPase/helicase.Panel A, hydrolysis of NTP by the SARS coronavirus NTPase/helicase versus NTP concentration versus NTP identity for eight different nucleotides. Data for the NTPase activity for each NTP over a range of NTP concentrations (0–3 mm) were fitted to a simple Michaelis-Menten model; the results are shown in Table I. Experimental conditions were: 25 μg/ml poly(U), 5 mm MgCl₂, 50 mm Tris-HCl, pH 6.6, 50-μl reaction volume, 0.4 pmol of helicase, 10-min reaction. Panel B, hydrolysis of dATP by the SARS coronavirus NTPase/helicase versus polynucleotide concentration for four different polynucleotides. Data are shown after subtraction of basal dATPase phosphate release. Data for the dATPase activity in the presence of each polynucleotide were fitted to a simple Michaelis-Menten model, with the fit shown by the four lines. Experimental conditions were: 1 mm dATP, 5 mm MgCl₂, 50 mm Tris-HCl, pH 6.6, 50-μl reaction volume, 0.7 pmol of SARS-CoV helicase, 10-min reaction.