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Article
Characterisation of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification
Peter McHugh
University of Oxford
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8679-4627
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3´-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.
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- FigureS1final.jpg
Nsp14-nsp10 is an RNA nuclease with a complex digestion pattern. (a) Size exclusion chromatogram for nsp14 alone, wild-type nsp14-nsp10 (designated nsp14- nsp10), and a control ‘nuclease-dead’ complex bearing alanine substitutions at residues D113 and E115 (nsp14D113A,E115A-nsp10). Traces of Superdex 200 16/60 runs are shown for Nsp14 alone and wild-type nsp14-nsp10 and trace of Superdex increase 10/300 run for the ‘nucleasedead’ complex. Wild-type nsp14-nsp10 complex elutes in a single peak at ~82.5 mL, Nsp14 alone at ~84.3 mL and ‘nuclease-dead’ complex at ~14.6 mL . SDS-PAGE analysis of the fraction across the peak confirms the presence of both nsp14 and nsp10 in fractions D1–D8 (see Suppl. Fig. 1e). (b) Intact mass spectrometry analysis of nsp14 alone, wild-type nsp14-nsp10 (designated nsp14- nsp10) and a control ‘nuclease-dead’ complex bearing substitutions at residues D113 and E115 (nsp14D113A,E115A-nsp10). LC-MS chromatogram shows the observed mass of all proteins is within range of the calculated mass. (c) Nsp14-nsp10 digests 20-mer and a 30-mer ssRNA substrates, but not a 10-mer ssRNA. (d) Nsp14-nsp10 is an RNA nuclease manifests a complex digestion pattern, digesting from the 3’ end in a single-nucleotide fashion until the 8th ribonucleotide, then cleaving at the 10th and 13th ribonucleotide. A single nucleotide ladder was used to determine the size of the released products. (e) SDS-PAGE of the fractions before, during and after the peak used from gel filtration for wildtype nsp14-nsp10. The elution peak (D1–D8) coincides precisely with the characteristic RNAse activity that we observe in our activity gel. The predicted molecular weight is 59 463 Da for nsp14 and 15 281 Da for nsp10. Increasing concentrations of protein (as indicated) were incubated with substrate at 37oC for 45 min, reactions were subsequently analysed by 20% denaturing PAGE to visualize product formation. The size of products was determined as shown in Suppl. Fig 1D. Main products are labelled *, ** and *** corresponding to 12-mer, 10-mer and 7-mer respectively. All oligos used are indicated in Suppl. table 1A and B. For Suppl. Figs. 1D and E, oligo 2 was used.
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Nsp14-nsp10 is metal-ion dependent RNA nuclease. (a) Nsp14-nsp10 activity is enhanced by the addition of both MgCl2 and MnCl2, but is inhibited by ZnCl2. We determined 5 mM MgCl2 as optimal for activity. (b) Consistent with its requirement for metal ions, nsp14-nsp10 activity is inhibited by the addition of metal ion chelators EDTA, EGTA, and ο-phenanthroline, with a particular sensitivity towards EDTA. 125 nM or 250 nM of protein was incubated with increasing concentrations of ions or chelators (as indicated) on ice for 15 min before adding substrate and incubating the reaction at 37oC for 45 min. Reactions were subsequently analysed by 20% denaturing PAGE to visualize product formation. For Suppl. Figs. 2A and B, oligo 2 was used.
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Nsp14-nsp10 activity is specific to RNA only and is able to process common chemical modifications on RNA. (a) Nsp14-nsp10 has no discernible nuclease activity on ssDNA or the DNA strand of an DNA:RNA hybrid. Interestingly, the complex is able to incise around an embedded ribonucleotide in a ssDNA oligo at concentrations above 125 nM. (b) Product formation (%) was quantified for Fig. 3 B comparing nsp14-nsp10 nuclease activity on dsRNA and RNA substrates containing termini and internal mismatches as outlined in Methods and Materials. All data are shown as mean ± s.e.m, and at least three biological replicates were used for each substrate. Nsp14-nsp10 shows no preference for mismatched oligonucleotides compared to dsRNA. mm: mismatch int. mm: internal mismatch (c) Nsp14-nsp10 is able process common chemical modifications of RNA, namely 2- methyladenine (m2A), 6-methyladenine (m6A) and inosine (I), both ssRNA and dsRNA with no apparent change compared to ssRNA or dsRNA. Increasing concentrations of protein (as indicated) were incubated with substrate at 37oC for 45 min, reactions were subsequently analysed by 20% denaturing PAGE to visualize product formation. The size of products was determined as shown in Suppl. Fig 1D. Main products are labelled *, ** and *** corresponding to 12-mer, 10-mer and 7-mer respectively. Oligomer substrates are in Suppl. Table 1A and B.
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Molecular docking of SARS-CoV nsp14-nsp10 using Autodock (a)&(b) Nsp14-nsp10 was docked with compounds within a grid box encompassing a surface focussed on the active. The calculated affinities of all binding modes are shown for each compound. Compounds are grouped based on their function groups (a), and by predicted inhibition activity (b); the dashed line describes the median of affinities for all compounds. (c) Nsp14-nsp10 was docked with compounds within a grid box encompassing the full surface. The affinities of binding modes were calculated for each compound and grouped based on predicted ???activities. The colouring reflects the location of binding mode on the structure of nsp14-nsp10; dashed line describes the median of affinities in this cohort. (d) The positions of highest-affinity docked poses as a function of the distance from the active site ExoN Mg2+ centre and the distance from the MTase substrate (GpppA) binding site. (e) Three representatives of predicted inhibitor binding locations on nsp14-nsp10 are shown: + (i) nsp-10 Zn site (disulfiram, purple), ExoN Mg2) (etoposide, salmon), and the MTase substrate binding site (A-1, cyan).
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Inhibition profile curves generated from gel-based nuclease assay data with the indicated compounds Inhibition profile curves were calculated by quantification of gel-based nuclease assay data as outlined in Methods and Materials. A decrease in the ‘% digested’ of the substrate represents an increase in inhibition. Data were plotted against log10 of [inhibitor] and dose response curves were generated using non-linear regression. Where possible IC50 values were obtained. All data are shown as mean ± sem; t least three biological replicates were used for each compound. In some cases an IC50 value was unable to be calculated, either due to incomplete inhibition, or the inability to fit a sigmoidal curve to the data.
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Synthesis scheme for N-hydroxyimide compounds A-1 to A-5 See Suppl. Materials and Methods for detailed synthesis protocols.
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The N-hydroxyimide-based compounds, A-1, AZ1353160 and AZ13623940 (AZ-B1), exhibit limited inhibition of nsp14-nsp10 Increasing concentrations (as indicated, in μM) of indicated compounds were incubated with 100 nM nsp14-nsp10 for 10 minutes at room temperature, before starting a standard nuclease assay reaction by the addition of ssRNA, incubating at 37oC for 45 min. Reaction products were analysed by 20% denaturing PAGE. A decrease in the generation of nucleolytic reaction products and a concomitant increase in undigested substrate indicates inhibition of nuclease activity at increasing inhibitor concentrations. - indicates no enzyme.
- FigureS8.jpg
Differential scanning spectrometry (DSF) of nsp14-nsp10 with drug and druglike compounds and N-hydroxyimide and hydroxypyrimidinone containing compounds (a) ΔTm of nsp14-nsp10 with drugs and drug-like compounds. Of all the compounds tested, only aurintricarboxylic acid (ATA), a known pan-nuclease inhibitor appears to aggregate the protein at high concentration (100 μM and 50 μM). (b) ΔTm of nsp14-nsp10 with N-hydroxyimide compounds Samples were heated from 25–95°C in 1°C per minute increments. Fluorescence intensities were plotted as a function of temperature to generate a sigmoidal curve with the inflection point of the transition curve indicating the melting temperature (Tm) of the protein. The Tm of nsp14-nsp10 is around 47°C. The ΔTm for each of the compound dilutions are shown as inset table. See Suppl. Materials and Methods for more detail
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List of RNA and DNA oligonucleotide sequences used to generate simple and complex RNA- and DNA-substrates (a) Each oligonucleotide sequence is numbered and a general description included. (b) To generate complex RNA and DNA structures, single-stranded oligos were annealed as designated by the numbers using the protocol described in the Materials and Methods.
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Table S1 continued
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List of compounds tested for in vitro inhibition on nsp14-nsp10 nuclease activity. Compound structures, functional groupings, and FDA approval status are shown alongside in vitro inhibition activity and AutoDock Vina scores of the highest-affinity binding modes for each compound. See Fig. 4, Fig. 5, Fig. 6, Suppl. Fig. 4, Suppl. Fig. 5, Suppl. Fig. 7, Suppl. Fig. 8 for relevant data.
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Article
Characterisation of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification
Peter McHugh
University of Oxford
Corresponding Author
ORCiD: https://orcid.org/0000-0002-8679-4627
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3´-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.