Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective
- Junior Research Group 2, Interdisciplinary Center for Clinical Research, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Germany
- Department of Biochemistry and Institute of Molecular Virology, University of Wisconsin-Madison, United States
- Architecture et Fonction des Macromolécules Biologiques, CNRS and Aix-Marseille Université, France
- Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, United States
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, United States
- The Ferrier Research Institute, Victoria University of Wellington, New Zealand
- Department of Microbiology and Immunology, University of North Carolina School of Medicine, United States
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, Institute for Protein Innovation, United States
- Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Institute for Human Infections and Immunity, University of Texas Medical Branch, Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, United States
- Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Netherlands
- Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, Netherlands
Figures
Figure 1 with 2 supplements
SARS-CoV-2 polymerase is a fast and processive RNA polymerase complex.
(A) Schematic of the magnetic tweezers assay to monitor RNA synthesis by the SARS-CoV-2 polymerase complex. A magnetic bead is attached to a glass coverslip surface by a 1043 long ssRNA construct …
Figure 1—figure supplement 1
Experimental conditions of SARS-CoV-2 polymerase high throughput magnetic tweezers experiments.
(A) SDS-PAGE analysis of SARS CoV-2 replicase proteins. Shown is a 15% polyacrylamide gel with 2 µg of purified nsp7 (9 kDa), nsp8 (22 kDa), and nsp12 (110 kDa). Broad-range molecular weight markers …
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Figure 1—figure supplement 1—source data 1
Source image for the SDS-PAGE gel in Figure 1—figure supplement 2A.
- https://cdn.elifesciences.org/articles/70968/elife-70968-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
Selection of SARS-CoV-2 polymerase elongation traces and reproducibility.
(A) SARS-CoV-2 polymerase activity traces acquired at 35 pN and 500 µM NTPs. Slow traces (<0.5% of the total number of events) are discarded from subsequent analysis. (B) Nucleotide addition rate …
Figure 2 with 4 supplements
3′-dATP is an effective chain terminator for the SARS-CoV-2 polymerase.
(A) SARS-CoV-2 replication traces for 500 µM NTPs and 500 µM 3′-dATP; and (D), for 50 µM ATP, 500 µM all other NTPs and 500 µM 3′-dATP. (B, E) SARS-CoV-2 polymerase product length for the 1043 nt …
Figure 2—figure supplement 1
Structure of the nucleotide analogs used in this study.
Figure 2—figure supplement 2
SARS-CoV-2 polymerase activity traces kinetics in presence of 3′-dATP.
(A) Dwell time distributions of SARS-CoV-2 polymerase activity traces acquired in the presence of 500 µM NTP either without (circles) or with 0.5 mM (triangles), 1 mM (squares), 1.5 mM (diamonds), …
Figure 2—figure supplement 3
Probabilistic model describing the competition for incorporation of a nucleic acid chain terminator NA and natural nucleotide.
From the empty active site state (E), either a terminator (T) or a natural nucleotide (N) can bind through direct competition with the first order binding rates and (solid arrows represent …
Figure 2—figure supplement 4
Decreasing the applied tension does not change the effect of nucleotide analogs (NAs) on the SARS-CoV-2 polymerase elongation.
All measurements are done at 25°C. (A) Product length of SARS-CoV-2 polymerase at 50 µM ATP and 500 µM all other NTPs (gray) or 300 µM NA and 50 µM of the competing NTP (purple: 3′-dATP, green: …
Figure 3 with 4 supplements
Remdesivir-TP (RDV-TP) is not a chain terminator but induces long-lived SARS-CoV-2 polymerase backtrack.
(A) SARS-CoV-2 polymerase activity traces for 500 µM NTPs and 100 µM RDV-TP. The inset is a zoom-in of the polymerase activity traces captured in the black square. (B) SARS-CoV-2 polymerase product …
Figure 3—figure supplement 1
SARS-CoV-2 polymerase elongation traces in presence of RDV-TP at 25°C.
(A) Examples of deep SARS-CoV-2 backtracks induced by RDV-TP incorporation (top) and traces showing no polymerase activity (bottom). Traces acquired using ultra-stable magnetic tweezers as described …
Figure 3—figure supplement 2
SARS-CoV-1 polymerase activity traces kinetics in presence of RDV-TP.
(A) SARS-CoV-1 polymerase activity traces for 500 µM NTPs and 100 µM RDV-TP. (B) SARS-CoV-1 polymerase product length for the 1043 nt long template using 500 µM NTPs, as a function of …
Figure 3—figure supplement 3
Lower ATP concentration at constant RDV-TP:ATP stoichiometry increases the effects of RDV-TP on SARS-CoV-2 polymerase elongation kinetics.
(A) SARS-CoV-2 polymerase activity traces for 10 µM RDV-TP with 50 µM ATP and 500 µM of the other NTPs. (B) Product length of SARS-CoV-2 polymerase at RDV-TP:ATP stoichiometry of 0/500, 100/500, …
Figure 3—figure supplement 4
SARS-CoV-2 polymerase activity traces kinetics in presence of RDV-TP at 37°C.
(A) Dwell time distributions of SARS-CoV-2 replication activity acquired in the presence of 500 µM NTP at 37°C without (gray) and with 100 µM RDV-TP (pink). The solid lines represent the fit of the …
Figure 4 with 1 supplement
T-1106-TP incorporation induces pauses of intermediate duration and backtrack.
(A) SARS-CoV-2 polymerase activity traces in the presence of 500 µM NTPs, in the presence of 500 µM T-1106-TP. The inset is a zoom-in of the polymerase activity traces captured in the black square. …
Figure 4—figure supplement 1
SARS-CoV-2 polymerase elongation in presence of T-1106-TP.
Using an ultra-stable magnetic tweezers configuration, we monitored pauses in SARS-CoV-2 polymerase activity traces at 58 Hz camera acquisition frequency (gray; 1 Hz low-pass filtered: dark gray), …
Figure 5 with 2 supplements
Sofosbuvir-TP is a poor SARS-CoV-2 polymerase inhibitor in contrast with 3′-dUTP.
(A, B) SARS-CoV-2 polymerase activity traces for 500 µM NTPs and 500 µM of either (A) Sofosbuvir-TP or (B) 3′-dUTP. (C, D) SARS-CoV-2 polymerase product length using the indicated concentration of …
Figure 5—figure supplement 1
SARS-CoV-2 polymerase activity traces kinetics in presence of Sofosbuvir-TP.
(A, E) SARS-CoV-2 replication time for the 1043 nt long template using the indicated concentration of UTP, 500 µM of other NTPs as a function of the stoichiometry of [Sofosbuvir-TP]/[UTP]. The …
Figure 5—figure supplement 2
SARS-CoV-2 polymerase activity traces kinetics in presence of 3′-dUTP.
(A, E) SARS-CoV-2 replication time for the 1043 nt long template using the indicated concentration of UTP, 500 µM of other NTPs as a function of the stoichiometry of [3′-dUTP]/[UTP]. The median …
Figure 6 with 4 supplements
ddhCTP and 3ʹ-dCTP inhibit efficiently the SARS-CoV-2 polymerase.
(A, B) SARS-CoV-2 polymerase activity traces for 500 µM NTPs and 500 µM of either (A) ddhCTP or (B) 3ʹ-dCTP. (C, D) SARS-CoV-2 polymerase product length using the indicated concentration of CTP, 500 …
Figure 6—figure supplement 1
SARS-CoV-2 polymerase activity traces in presence of ddhCTP.
(A, E) SARS-CoV-2 replication time for the 1043 nt long template using the indicated concentration of CTP, 500 µM of other NTPs, and the indicated stoichiometry of [ddhCTP]/[CTP]. The median values …
Figure 6—figure supplement 2
SARS-CoV-2 polymerase activity traces kinetics in presence of 3′-dCTP.
(A, E) SARS-CoV-2 replication time for the 1043 nt long template using the indicated concentration of CTP, 500 µM of other NTPs as a function of the stoichiometry of [3′-dCTP]/[CTP]. The median …
Figure 6—figure supplement 3
ddhC does not inhibit SARS-CoV-2 replication in huh7-hACE2 cells.
Huh7-hACE2 cells in 96-well were incubated with the indicated concentrations of the tested compounds for 1.5 hr before SARS-CoV-2 (USA-WA1/2020 isolate) was added at MOI of 0.05. At ~24 hpi, the …
Figure 6—figure supplement 4
SARS-CoV-2 nsp14 exoribonuclease knockout is not replicative.
(A) SARS-CoV-2 genome. The SARS-CoV-2 nsp14 exoribonuclease nucleotides and amino acid mutations (D90A/E92A) are indicated. (B) Phase-contrast images of electroporated cells. Vero E6 cells were …
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Figure 6—figure supplement 4—source data 1
Source image for the agarose gel in Figure 6—figure supplement 4D.
- https://cdn.elifesciences.org/articles/70968/elife-70968-fig6-figsupp4-data1-v1.zip
Tables
Table 1
Summary table for the investigated NAs.
| Modification | Incorporation pathway | Mechanism of action | Main conclusions | ||
|---|---|---|---|---|---|
| In vitro incorporation efficiency | In vivo efficacy | ||||
| 3′-dATP | Ribose, 3′ | NAB | Chain terminator | Medium | Unreported |
| 3′-dCTP | Ribose, 3′ | NAB | Chain terminator | Medium | Unreported |
| 3′-dUTP | Ribose, 3′ | NAB | Chain terminator | Medium | Unreported |
| Remdesivir-TP | Ribose, 1′ | SNA, VSNA | Polymerase backtrack | Very high | Very high |
| T-1106-TP | Base | VSNA | Induces pauses (mutagenic) | Medium | Unreported |
| Sofosbuvir-TP | Ribose, 2′ | NAB | Chain terminator | Very low | None |
| ddhCTP | Ribose, 3′ | NAB | Chain terminator | low | None |
Additional files
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Supplementary file 1
Summary of statistics, rates, and probabilities for each experimental condition presented in this study.
- https://cdn.elifesciences.org/articles/70968/elife-70968-supp1-v1.xls
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Supplementary file 2
Summary of statistics, rates, and probabilities for each experimental condition presented in this study.
- https://cdn.elifesciences.org/articles/70968/elife-70968-supp2-v1.xls
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70968/elife-70968-transrepform-v1.docx
