Inhibition of sars-cov-2 polymerase by nucleotide analogs from a single-molecule perspective

Mona Seifert; Subhas C. Bera; Pauline Van Nies; Robert N. Kirchdoerfer; Ashleigh Shannon; Thi Tuyet Nhung Le; Xiangzhi Meng; Hongjie Xia; James M. Wood; Lawrence D. Harris; Flavia S. Papini; Jamie J. Arnold; Steven Almo; Tyler L. Grove; Pei Yong Shi; Yan Xiang; Bruno Canard; Martin Depken; Craig E. Cameron; David Dulin

doi:10.7554/eLife.70968

Inhibition of sars-cov-2 polymerase by nucleotide analogs from a single-molecule perspective

Mona Seifert, Subhas C. Bera, Pauline Van Nies, Robert N. Kirchdoerfer, Ashleigh Shannon, Thi Tuyet Nhung Le, Xiangzhi Meng, Hongjie Xia, James M. Wood, Lawrence D. Harris, Flavia S. Papini, Jamie J. Arnold, Steven Almo, Tyler L. Grove, Pei Yong Shi, Yan Xiang, Bruno Canard, Martin Depken, Craig E. Cameron, David Dulin^*

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

The absence of ‘shovel-ready’ anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

Original language	English
Article number	e70968
Pages (from-to)	1-26
Number of pages	26
Journal	eLife
Volume	2021
Issue number	10
Early online date	7 Oct 2021
DOIs	https://doi.org/10.7554/eLife.70968
Publication status	Published - Oct 2021

Bibliographical note

Funding Information:
National Institutes of HealthAI123498.

Funding Information:
The authors thank Joy Feng from Gilead Sciences for providing RDV-TP, and Veronique Fattorini and Barbara Selisko for excellent technical assistance and help in SARS-CoV-1 proteins purification. DD was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation grant DFG-DU-1872/ 3-1 and BaSyC – Building a Synthetic Cell’ Gravitation grant (024.003.019) of the Netherlands Ministry of Education, Culture and Science (OCW) and the Netherlands Organisation for Scientific Research (NWO). DD thanks OICE for providing office and lab space, and access to their molecular biology lab. RNK was supported by grant AI123498 from NIAID, NIH. JMW and LDH thank the Ministry of Business Innovation and Employment Contract UOOX1904 (NZ). YX was supported by the National Institutes of Health (NIH) grant AI151638. SARS-Related Coronavirus 2, Isolate USA-WA1/ 2020 (NR-52281) was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. PYS was supported by NIH grants AI134907 and UL1TR001439, and awards from the Sealy and Smith Foundation, Kleberg Foundation, the John S Dunn Foundation, the Amon G. Carter Foundation, the Gilson Longenbaugh Foundation, and the Summerfield Robert Foundation. JJA and CEC were supported by grant AI045818 from NIAID, NIH. AS, TTNL, and BC acknowledge grants by the Fondation pour la Recherche Médicale (Aide aux équipes), the SCORE project H2020 SC1-PHE-Coronavirus-2020 (grant#101003627), and the REACTing initiative (REsearch and ACTion targeting emerging infectious diseases).

Publisher Copyright:
© Seifert et al.

Funding

National Institutes of HealthAI123498. The authors thank Joy Feng from Gilead Sciences for providing RDV-TP, and Veronique Fattorini and Barbara Selisko for excellent technical assistance and help in SARS-CoV-1 proteins purification. DD was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation grant DFG-DU-1872/ 3-1 and BaSyC – Building a Synthetic Cell’ Gravitation grant (024.003.019) of the Netherlands Ministry of Education, Culture and Science (OCW) and the Netherlands Organisation for Scientific Research (NWO). DD thanks OICE for providing office and lab space, and access to their molecular biology lab. RNK was supported by grant AI123498 from NIAID, NIH. JMW and LDH thank the Ministry of Business Innovation and Employment Contract UOOX1904 (NZ). YX was supported by the National Institutes of Health (NIH) grant AI151638. SARS-Related Coronavirus 2, Isolate USA-WA1/ 2020 (NR-52281) was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. PYS was supported by NIH grants AI134907 and UL1TR001439, and awards from the Sealy and Smith Foundation, Kleberg Foundation, the John S Dunn Foundation, the Amon G. Carter Foundation, the Gilson Longenbaugh Foundation, and the Summerfield Robert Foundation. JJA and CEC were supported by grant AI045818 from NIAID, NIH. AS, TTNL, and BC acknowledge grants by the Fondation pour la Recherche Médicale (Aide aux équipes), the SCORE project H2020 SC1-PHE-Coronavirus-2020 (grant#101003627), and the REACTing initiative (REsearch and ACTion targeting emerging infectious diseases).

Funders	Funder number
H2020 SC1-PHE-Coronavirus-2020	101003627
National Institutes of HealthAI123498
Smith Foundation
University of Erlangen-Nuremberg
National Institutes of Health	AI134907, AI151638
National Institute of Allergy and Infectious Diseases
Amon G. Carter Foundation
National Center for Advancing Translational Sciences	UL1TR001439
John S. Dunn Foundation
Gillson Longenbaugh Foundation
Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation
Summerfield G. Roberts Foundation	AI045818
Deutsche Forschungsgemeinschaft	DFG-DU-1872/ 3-1, 024.003.019
Fondation pour la Recherche Médicale
Ministerie van Onderwijs, Cultuur en Wetenschap
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	AI123498
Ministry of Business, Innovation and Employment	UOOX1904
Interdisziplinäres Zentrum für Klinische Forschung, Universitätsklinikum Würzburg

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Seifert, M., Bera, S. C., Van Nies, P., Kirchdoerfer, R. N., Shannon, A., Le, T. T. N., Meng, X., Xia, H., Wood, J. M., Harris, L. D., Papini, F. S., Arnold, J. J., Almo, S., Grove, T. L., Shi, P. Y., Xiang, Y., Canard, B., Depken, M., Cameron, C. E., & Dulin, D. (2021). Inhibition of sars-cov-2 polymerase by nucleotide analogs from a single-molecule perspective. eLife, 2021(10), 1-26. Article e70968. https://doi.org/10.7554/eLife.70968

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title = "Inhibition of sars-cov-2 polymerase by nucleotide analogs from a single-molecule perspective",

abstract = "The absence of {\textquoteleft}shovel-ready{\textquoteright} anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.",

author = "Mona Seifert and Bera, {Subhas C.} and {Van Nies}, Pauline and Kirchdoerfer, {Robert N.} and Ashleigh Shannon and Le, {Thi Tuyet Nhung} and Xiangzhi Meng and Hongjie Xia and Wood, {James M.} and Harris, {Lawrence D.} and Papini, {Flavia S.} and Arnold, {Jamie J.} and Steven Almo and Grove, {Tyler L.} and Shi, {Pei Yong} and Yan Xiang and Bruno Canard and Martin Depken and Cameron, {Craig E.} and David Dulin",

note = "Funding Information: National Institutes of HealthAI123498. Funding Information: The authors thank Joy Feng from Gilead Sciences for providing RDV-TP, and Veronique Fattorini and Barbara Selisko for excellent technical assistance and help in SARS-CoV-1 proteins purification. DD was supported by the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation grant DFG-DU-1872/ 3-1 and BaSyC – Building a Synthetic Cell{\textquoteright} Gravitation grant (024.003.019) of the Netherlands Ministry of Education, Culture and Science (OCW) and the Netherlands Organisation for Scientific Research (NWO). DD thanks OICE for providing office and lab space, and access to their molecular biology lab. RNK was supported by grant AI123498 from NIAID, NIH. JMW and LDH thank the Ministry of Business Innovation and Employment Contract UOOX1904 (NZ). YX was supported by the National Institutes of Health (NIH) grant AI151638. SARS-Related Coronavirus 2, Isolate USA-WA1/ 2020 (NR-52281) was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH. PYS was supported by NIH grants AI134907 and UL1TR001439, and awards from the Sealy and Smith Foundation, Kleberg Foundation, the John S Dunn Foundation, the Amon G. Carter Foundation, the Gilson Longenbaugh Foundation, and the Summerfield Robert Foundation. JJA and CEC were supported by grant AI045818 from NIAID, NIH. AS, TTNL, and BC acknowledge grants by the Fondation pour la Recherche M{\'e}dicale (Aide aux {\'e}quipes), the SCORE project H2020 SC1-PHE-Coronavirus-2020 (grant#101003627), and the REACTing initiative (REsearch and ACTion targeting emerging infectious diseases). Publisher Copyright: {\textcopyright} Seifert et al.",

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Seifert, M, Bera, SC, Van Nies, P, Kirchdoerfer, RN, Shannon, A, Le, TTN, Meng, X, Xia, H, Wood, JM, Harris, LD, Papini, FS, Arnold, JJ, Almo, S, Grove, TL, Shi, PY, Xiang, Y, Canard, B, Depken, M, Cameron, CE & Dulin, D 2021, 'Inhibition of sars-cov-2 polymerase by nucleotide analogs from a single-molecule perspective', eLife, vol. 2021, no. 10, e70968, pp. 1-26. https://doi.org/10.7554/eLife.70968

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AU - Shannon, Ashleigh

AU - Le, Thi Tuyet Nhung

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AU - Xiang, Yan

AU - Canard, Bruno

AU - Depken, Martin

AU - Cameron, Craig E.

AU - Dulin, David

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Inhibition of sars-cov-2 polymerase by nucleotide analogs from a single-molecule perspective

Abstract

Bibliographical note

Funding

UN SDGs

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