Mechanism of nucleic acid unwinding by SARS-CoV helicase

Abstract

The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5' → 3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. To elucidate the nature of the intermediates we carried out transient kinetic analysis of the nsp13 helicase activity. We demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base-pairs (bp) each, with a catalytic rate of 30 steps per second. Therefore the net unwinding rate is ~280 base-pairs per second. We also showed that nsp12, the SARS-CoV RNA-dependent RNA polymerase (RdRp), enhances (2-fold) the catalytic efficiency of nsp13 by increasing the step size of nucleic acid (RNA/RNA or DNA/DNA) unwinding. This effect is specific for SARS-CoV nsp12, as no change in nsp13 activity was observed when foot-and-mouth-disease virus RdRp was used in place of nsp12. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis.

Figure 1. Comparison of unwinding activity of three nsp13 variants.

Comparison of the helicase activity at varying time points for GST-nsp13 (Glutathione S-Transferase-tagged nsp13), MBP-nsp13 (Maltose Binding Protein-tagged nsp13), and H₆-nsp13 (hexahistidine-tagged nsp13) using 100 nM of each enzyme and 5 nM 60/40-mer (30ss:30ds) as substrate at 30°C. The products were separated and analyzed by 6% non-denaturing PAGE. Time points for the GST-nsp13-catalyzed unwinding experiments are more than 100 times smaller than for the MBP-nsp13- and H₆-nsp13-catalyzed experiments (0.01–5 vs. 5–600 seconds).

Figure 2. Hydrolysis of ATP during unwinding of dsDNA by GST-nsp13 and H₆-nsp13.

GST-nsp13 or H₆-nsp13 was pre-incubated with 31/18-mer and rapidly mixed with various concentrations of γ^-32P-ATP (2 µM, 5 µM, 12 µM, 25 µM and 50 µM,) for various reaction times (0.005–0.5 s for GST nsp13 and 0.005–5 s for H₆-nsp13). The reaction products were separated by thin-layer chromatography and visualized by autoradiography. ATP hydrolysis was quantitated as amount of Pi released and plotted against reaction time. The data were fit to a single exponential equation to calculate initial hydrolysis rates. The derived rates were plotted against ATP concentration and the data points were fit to a hyperbolic function to determine the (A) GST-nsp13 optimal ATP hydrolysis rate k_hydro(ATP) 104.1±4. s⁻¹ and (B) H₆-nsp13 optimal ATP hydrolysis rate k_hydro(ATP) 0.2±0.006 s⁻¹ (two independent experiments).

Figure 3. Substrate specificity of nsp13.

(A) Comparison of helicase activity of GST-nsp13 using same sequence of dsDNA (♦) or dsRNA (▪) 38/18-mer substrates under conditions described in the methods section. B) Four different substrates were designed as shown in Supplementary Fig. 1 (forked substrate, 31/18-mer, 48/18-mer, and 55/18-mer). The helicase activity of 100 nM nsp13 was measured with 5 nM of each of the substrates at 30°C for 5 secs and the products were separated on a non-denaturing 6% polyacrylamide gel and visualized using a PhosphorImager. C) Four different substrates with 5′ overhang lengths varying from 5 to 20 nucleotides were designed to determine the minimum length of loading strand required by nsp13 to efficiently unwind its substrate. The helicase activity of nsp13 (10 nM) was assessed on these substrates (5 nM each) at 30°C for 10 mins and the products were separated on a non-denaturing 6% polyacrylamide gel and visualized using the PhosphorImager.

Figure 4. GST-nsp13-catalyzed unwinding of dsDNA of varying lengths, performed under single-turnover conditions.

(A) The minimal kinetic mechanism of nucleic acid unwinding by nsp13. The minimal reaction scheme describing a series of n sequential steps of helicase-catalyzed translocation and unwinding. H-DNA, Helicase-DNA complex; I, intermediate; k _np is a macroscopic rate constant for conversion of nonproductive (H-DNA)_NP to productive Helicase-DNA complexes (H-DNA)_L. Each identical step in the reaction pathway is defined by a forward rate constant, k_u, and a dissociation rate constant, k_d. (B) Time course of nsp13-catalyzed unwinding of dsDNA. The double stranded component of the substrates was: 18 bp (•), 30 bp (▪), 40 bp (▴), 60 bp (♦). Experiments were repeated at least twice for each substrate. (C) Plot of the amplitude of nucleic acid unwinding as a function of the number of kinetic steps determined for each substrate (for substrates with dsDNA component of 18, 30, 40, and 60 bp). Data were fit to the equation A = Pⁿ and the processivity, P, of nsp13 was estimated from the amplitude A and number of steps n and found to be 0.80±0.03.

Figure 5. Effect of nsp12 on the unwinding activity of nsp13.

Time course of DNA (5 nM. 80/60-mer) unwinding by 100 nM GST-nsp13 in the presence of 0 nM nsp12H₆ (▴), 250 nM nsp12H₆ (▪) and 500 nM nsp12H₆ (▾). The products were separated and analyzed by 6% non-denaturing PAGE and the fraction of DNA unwound was plotted against reaction time. The experimental data were fit globally to equation (1) (Materials and Methods-Analyses of DNA unwinding) derived from Scheme 1 (Fig. 4A).

Figure 6. Comparison of the effect of nsp12 on nsp13 activity using RNA and DNA substrates.

(A, B): Time course of (A) RNA (31/18-mer) and (B) DNA (31/18-mer) unwinding by GST-nsp13 alone (▪), or by GST-nsp13 with nsp12 (♦) or GST-nsp13 with Foot-and-Mouth Disease Virus RNA polymerase (▴). (C, D) Time course of (C) RNA (31/18-mer) and (D) DNA (31/18-mer) unwinding by H₆-nsp13 alone (•), or by H₆-nsp13 with nsp12 (▪) or by H₆-nsp13 with Glutathione-S-transferase protein (without the nsp13 part) (▾).