Two opposite abilities of the infectious bronchitis virus helicase Nsp13: separating the duplex and promoting the annealing of single-stranded nucleic acid

Figures

• 

  Figure 1. Expression and identification of IBV Nsp13. (A) Recombinant plasmid pET-28a-Nsp13. Lane 1: plasmid was digested by double restriction enzymes, lane 2: recombinant plasmid. (B) Induced expression of protein Nsp13. Lane 1: uninduced bacterial fluid, lanes 2–6: bacterial fluid after induction. (C) Recombinant IBV Nsp13 was analyzed by electrophoresis on a 10% SDS-PAGE gel and stained with Coomassie brilliant blue. (D) Nsp13 was analyzed by Western blotting with anti-His antibody. (E) Validation of Nsp13 activity by the unwinding reaction. The arrows and asterisks in the figures indicate the target proteins NSP13 and 5′-FAM, respectively.

• 

  Figure 2. Detection of the ATPase activity of IBV Nsp13. (A) Schematic diagram of the principle of the ATPase assay. (B) Determination of the ATPase activity at different concentrations of Nsp13. Purified Nsp13 (0, 1, 2.5, 5, 12.5, 25, 75, 150, and 200 nM) were incubated with 5 μM ATP in reaction buffer and then added kinase-lumi reagent, and the bioluminescence was measured. (C) The ATPase activity of Nsp13 at the appointed time (0, 0.5, 1, 2, 4, 8, 16, and 32 min) was measured. (D) Determination of the ATPase activity of Nsp13 in the presence of 3 mM MgCl2, CaCl2, ZnCl2, and MnCl2. (E) Determination of the ATPase activity of Nsp13 in the presence of various concentrations of MgCl2 (0, 2, 4, 6, and 8 mM). Error bars in figures represent the standard deviation.

• 

  Figure 3. Unwinding polarity of IBV helicase Nsp13. (A) Structures of duplex substrates with 5′-overhang, 3′-overhang, and blunt end. (B) dsRNA with different structures was unwound by Nsp13. (C) dsDNA with different structures was unwound by Nsp13. The asterisk in figures denotes the fluorophore 5′-FAM.

• 

  Figure 4. Determination of the factors that regulate the unwinding activity of IBV helicase Nsp13. (A) Assessment of the effect of NTP species on the unwinding activity of Nsp13. (B) Assessment of the effect of ATP concentration on the unwinding activity of Nsp13. (C) Assessment of the effect of metal ions on the unwinding activity of Nsp13. (D) Assessment of the effect of pH on the unwinding activity of Nsp13. (E) Assessment of the effect of helicase concentration on the unwinding activity of Nsp13. Filled triangles represent the positive controls with heat-denatured substrates, and the unwinding reactions without Nsp13 were the negative controls. The asterisks in the figures indicate the 5′-FAM. The error bars in the figures represent the standard deviation.

• 

  Figure 5. Effect of polyglycol modification of the substrate backbone on the Nsp13-catalyzed unwinding reaction. (A) Substrates containing the PEG linker in the top or bottom strand were unwound by Nsp13. (B) The reaction products were separated on native PAGE gels. Filled triangles represent the positive controls with heat-denatured substrates, and the unwinding reactions without Nsp13 were the negative controls. (C) The derived fractions of RNA unwound at indicated time points were plotted against time. (D) The unwinding amplitudes of substrates with various polyglycol modifications. The asterisks in the figures indicate the 5′-FAM. Error bars in figures represent the standard deviation.

• 

  Figure 6. Determination of the annealing activity of IBV Nsp13. (A) Spontaneous annealing of ssRNA was detected in the reaction. (B) Annealing reaction of single-stranded RNA was catalyzed by Nsp13 in the absence of ATP. (C) Annealing reaction of single-stranded RNA was catalyzed by Nsp13 in the presence of ATP. (D) Annealing reaction of single-stranded RNA was catalyzed by inactive Nsp13. (E) The percentage of dsRNA formed in the annealing reaction was plotted against time to show the progress of product formation. (F) The amplitudes of the annealing reaction under various conditions. The asterisks in the figures indicate the 5′-FAM. Error bars in figures represent the standard deviation.

• 

  Figure 7. Scheme of the catalytic activities of IBV helicase Nsp13. Nsp13 unwinds duplex substrates with 5′-overhang by utilizing the energy derived from ATP hydrolysis and promotes the annealing of complementary single strands in the absence of ATP. The asterisks in the figures indicate the 5′-FAM.