SARS-Coronavirus-2 Nsp13 Possesses NTPase and RNA Helicase Activities That Can Be Inhibited by Bismuth Salts

Abstract

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.

Keywords: Antiviral target; Helicase; NTPase; Nsp13; SARS-coronavirus-2 (SARS-CoV-2).

Fig. 1

SARS-CoV-2 nsp13 has NTPase activity. A 10 pmol/L MBP-nsp13 was reacted with the indicated NTPs (2.5 mmol/L for each). The NTPase activity was measured as nanomoles of released inorganic phosphate (pi) by using a sensitive colorimetric assay. The reaction without any NTP was used as negative control (None). B 2.5 mmol/L ATP was incubated with MBP alone or MBP-nsp13 at the increasing concentrations. C 10 pmol/L MBP-nsp13 was reacted with 2.5 mmol/L ATP at 2 mmol/L indicated divalent metal ions. The reaction without any divalent metal ion was used as negative control (None). D 10 pmol/L MBP-nsp13 was reacted with 2.5 mmol/L ATP at the indicated concentrations of MgCl₂. MBP alone was used as the negative control. Error bars represent standard deviation (SD) values from three separate experiments.

Fig. 2

SARS-CoV-2 nsp13 has RNA helix unwinding activity. A Schematic illustration of the RNA helix substrate (R/R*). Asterisks indicate the HEX-labelled strands. B The RNA helix substrate (0.1 pmol/L) was reacted with each indicated protein (20 pmol/L). And the unwinding activity was assessed via gel electrophoresis and scanning on a Typhoon 9500 imager. Non-boiled reaction mixture (lane 1) and reaction mixture with MBP alone (lane 3) were used as negative controls, and boiled reaction mixture (lane 2) was used as positive control. C The RNA helix unwinding assay was performed in the presence of increasing concentrations of MBP-nsp13. D MBP-nsp13 (20 pmol/L) was reacted with the RNA helix substrate (0.1 pmol/L) in reaction mixture at different reaction time.

Fig. 3

Optimal biochemical reaction conditions for the RNA helix unwinding activity of SARS-CoV-2 nsp13. A Schematic illustration of the RNA helix substrate (R/R*). Asterisks indicate the HEX-labelled strands. B–D MBP-nsp13 (20 pmol/L) was reacted with the RNA helix substrate (0.1 pmol/L) in the presence of the indicated NTPs (5 mmol/L) (B) each indicated divalent metal ion (2 mmol/L for each) (C), increasing concentrations of MgCl₂ (D).

Fig. 4

SARS-CoV-2 nsp13 unwinds RNA helix in the 5′–3′ directionality. A–C Schematic illustrations of the RNA helix substrates with 5′-tailed (A), 3′-tailed (B), and blunt ends (C). Asterisks indicate the HEX-labelled strand. D MBP-nsp13 (20 pmol/L) was reacted with 0.1 pmol/L 5′-tailed (lane 3) or 3′-tailed (lane 6) RNA helix substrate. E MBP-nsp13 (20 pmol/L) was reacted with 0.1 pmol/L helix substrate with blunt ends.

Fig. 5

Bismuth salts inhibit the ATPase and RNA helix unwinding activities of SARS-CoV-2 nsp13. A The ATPase activity of MBP-nsp13 (10 pmol/L) in the presence of the indicated bismuth salts (10 μmol/L for each). X-axis were expressed as the different bismuth salts. Values (Y-axis) were expressed as percentages of those of mock-treated (−) ATPase activity of MBP-nsp13 (10 pmol/L). The error bars represent SD values from three separate experiments. B Upper panel: schematic illustration of the RNA helix substrate (R/R*); asterisks indicate the HEX-labelled strand. Lower panel: The RNA helix unwinding assays were performed by incubating 0.1 pmol/L RNA helix substrate with MBP-nsp13 (2 pmol/L) in presence of the indicated bismuth salts (10 μmol/L for each).

Fig. 6

BPC and RBC inhibit the ATPase and RNA helix unwinding activities of SARS-CoV-2 nsp13 in a dose-dependent manner. A and D The ATPase activity of MBP-nsp13 (10 pmol/L) was performed in the presence of increasing concentrations of BPC (A) or RBC (D) as indicated. B and E The RNA helix unwinding activity of MBP-nsp13 (2 pmol/L) was performed in the presence of increasing concentrations of BPC (B) or RBC (E) as indicated. C and F The unwinding activities in (B and E) were plotted as percentages of the released HEX-labelled RNA from the total RNA helix (Y-axis) at indicated concentrations (X-axis) of BPC (C) or RBC (F). The error bars represent SD values from three separate experiments.