SARS-CoV-2 Nucleocapsid Protein Has DNA-Melting and Strand-Annealing Activities With Different Properties From SARS-CoV-2 Nsp13

Abstract

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world and has had a devastating impact on health and economy. The biochemical characterization of SARS-CoV-2 proteins is important for drug design and development. In this study, we discovered that the SARS-CoV-2 nucleocapsid protein can melt double-stranded DNA (dsDNA) in the 5'-3' direction, similar to SARS-CoV-2 nonstructural protein 13. However, the unwinding activity of SARS-CoV-2 nucleocapsid protein was found to be more than 22 times weaker than that of SARS-CoV-2 nonstructural protein 13, and the melting process was independent of nucleoside triphosphates and Mg²⁺. Interestingly, at low concentrations, the SARS-CoV-2 nucleocapsid protein exhibited a stronger annealing activity than SARS-CoV-2 nonstructural protein 13; however, at high concentrations, it promoted the melting of dsDNA. These findings have deepened our understanding of the SARS-CoV-2 nucleocapsid protein and will help provide novel insights into antiviral drug development.

Keywords: Nsp13; SARS-CoV-2; helicase; nucleocapsid protein; unwinding.

Figure 1

SDS-PAGE analysis of purified CoV-2 N and CoV-2 Nsp13 preparations. (A,B) Approximately 25 μg of purified CoV-2 N and CoV-2 Nsp13 were electrophoresed on 10% polyacrylamide gels and stained with Coomassie Brilliant Blue. The positions of the molecular weight (MW) markers are shown on the left of each image. The molecular weights of CoV-2 N and CoV-2 Nsp13 were approximately 47 kDa and 68 kDa, respectively. Lane N: CoV-2 N; Lane Nsp13: CoV-2 Nsp13.

Figure 2

Unwinding polarity of CoV-2 N and CoV-2 Nsp13. (A,B) CoV-2 N and CoV-2 Nsp13 unwind dsDNA containing a 5′-overhang (5′-OhS22D21). (C,D) CoV-2 N and CoV-2 Nsp13 cannot unwind dsDNA with a 3′-overhang (3’-OhS22D21). All experiments were performed under standard experimental conditions. dsDNA (5′-OhS22D21 or 3’-OhS22D21) was assayed at 10 nM and contained a fluorescent label on the 3′ end, and protein concentration gradually increased. Lane ds: duplex -DNA; Lane ss: single DNA with 3′-fluorescein (3’-FAM; shown in Table 1). Top of the arrow: duplex DNA is not unwound; Bottom of the arrow: unwound single-stranded DNA. Native PAGE (Acr:Bis = 39:1; 12%), 100 V, 80 min. The fluorescence signals were visualized using a ChemiDoc MP Imaging System (Bio-Rad, California, United States). The detailed experimental conditions for the unwinding assay are described in the Materials and Methods section.

Figure 3

Determination and comparison of optimal buffer compositions for CoV-2 N and CoV-2 Nsp13 unwinding. (A–F) Protein concentrations of CoV-2 N and CoV-2 Nsp13 were 3 μM (note: the protein concentration is 5 μM in A) and 0.3 μM (note: the protein concentration is 0.1 μM in F), respectively, and the reactions were performed in buffer A and buffer B (removing the single-factor NaCl, Mg²⁺, DTT shown in A–F). (G–J) The protein concentrations of CoV-2 N and CoV-2 Nsp13 were saturating concentrations of 5 μM and 0.3 μM, respectively. All solutions used were kept on ice for precooling. Samples were placed on ice before reactions were initiated. A water bath of the corresponding temperature was used immediately after reactions were started, and stop buffer was added at appropriate times to terminate reactions. All used substrates were 5′-overhang DNA (5’-OhS22D21) at 10 nM.

Figure 4

Unwinding activities of CoV-2 N and CoV-2 Nsp13 in the presence of different nucleoside triphosphates at different concentrations. (A,B) The 5′-overhang DNA substrate at 10 nM (5’-OhS22D21). CoV-2 N concentration was 3 μM in buffer A and CoV-2 Nsp13 was 0.3 μM in buffer B, respectively. For a clearer analysis of the propensity of CoV-2 Nsp13 to NTP species, we performed comparisons by scanning gray values using Image Lab software (Supplementary Figure 4). All experiments were performed under experimental conditions described in “Materials and Methods.”

Figure 5

Comparison of the effect of single-strand overhang length on the unwinding ability of CoV-2 N and CoV-2 Nsp13. (A,B) Blunt-end dsDNA substrate (DS32). (C,D) Comparison of the effect of single-strand overhang length (4 nt–16 nt) on the unwinding activities of CoV-2 N and CoV-2 Nsp13. (c1, d1) 5’-OhS4D20; (c2, d2) 5’-OhS12D20; (c3, d3) 5’-OhS14D20; (c4, d4) 5’-OhS15D20; (c5, d5) 5’-OhS16D20. The unwinding activities of CoV-2 N and CoV-2 Nsp13 were assayed in the presence of increasing protein concentrations in buffer A and buffer B, respectively. All 5′-overhang dsDNA substrates used at 10 nM.

Figure 6

Comparison of the effect of the dsDNA bubble structure on the unwinding ability of CoV-2 N and CoV-2 Nsp13. (a1, b1) BS4; (a2, b2) BS12; (a3, b3) BS14; (a4, b4) BS15; and (a5, b5) BS16. All experiments were performed using a series of dsDNA bubble substrates (shown in Table 1), The unwinding activities of CoV-2 N and CoV-2 Nsp13 were assayed in the presence of increasing protein concentrations in buffer A and buffer B, respectively. All the bubble dsDNA substrates fixed at 10 nM. All experiments were performed under standard experimental conditions as described in “Materials and Methods.” (A,B) Comparison of dsDNA bubble structure (4 nt -16 nt) on the unwinding activities of CoV-2 N and CoV-2 Nsp13.

Figure 7

Binding and annealing activities of CoV-2 N and CoV-2 Nsp13. (A,B) The binding activity of single DNA FAM-labeled (S43) to CoV-2 N and CoV-2 Nsp13 was evaluated by comparing binding fractions and dissociation constants (Kd). The substrate used was ssDNA-S43 at a concentration of 5 nM, and protein concentration(s) gradually increased. All experiments were performed under standard experimental conditions as described in the Materials and Methods. (C,D) The annealing activities of CoV-2 N (C) and CoV-2 Nsp13 (D) were assayed in the presence of increasing protein concentrations in buffer A and buffer B at 30°C for 10 min, respectively. Native PAGE (Acr:Bis = 39:1) 12%, 100 V, 80 min. The two single-stranded DNA substrates (S43, S21) were used at 10 nM. Fluorescence signals were visualized using a ChemiDoc MP Imaging System (Bio-Rad, California, United States). Lane ss: single DNA with 3′-fluorescein (S43; shown in Table 1). Top of the arrow: annealed double-stranded DNA; bottom of the arrow: unannealed free ssDNA(S43). The results of (C,D) were visualized by a ChemiDoc MP Imaging System (Bio-Rad, California, United States).

Figure 8

(A) The dynamic self-dissociation of dsDNA. (B) The dsDNA melting model of CoV-2 N-protein.