Interfering with nucleotide excision by the coronavirus 3'-to-5' exoribonuclease

Rukesh Chinthapatla; Mohamad Sotoudegan; Pankaj Srivastava; Thomas K. Anderson; Ibrahim M. Moustafa; Kellan T. Passow; Samantha A. Kennelly; Ramkumar Moorthy; David Dulin; Joy Y. Feng; Daniel A. Harki; Robert N. Kirchdoerfer; Craig E. Cameron; Jamie J. Arnold

doi:10.1093/nar/gkac1177

Interfering with nucleotide excision by the coronavirus 3^'-to-5^' exoribonuclease

Rukesh Chinthapatla, Mohamad Sotoudegan, Pankaj Srivastava, Thomas K. Anderson, Ibrahim M. Moustafa, Kellan T. Passow, Samantha A. Kennelly, Ramkumar Moorthy, David Dulin, Joy Y. Feng, Daniel A. Harki, Robert N. Kirchdoerfer, Craig E. Cameron, Jamie J. Arnold

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3^'-to-5^' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3^'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3^'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.

Original language	English (US)
Pages (from-to)	315-336
Number of pages	22
Journal	Nucleic acids research
Volume	51
Issue number	1
DOIs	https://doi.org/10.1093/nar/gkac1177
State	Published - Jan 11 2023

Bibliographical note

Funding Information:
Our thanks to Roel Fleuren and Efra Rivera-Serrano for their contributions to the production of figures and models. We thank Stewart Shuman for providing the expression plasmid for RtcA. National Institutes of Health [R01AI161841 to C.E.C., J.J.A., D.D.; P01CA234228, R01GM110129 to D.A.H., R01AI158463 to R.K.]; University of Minnesota, Office of the Vice President for Research, Grant-in-Aid (to D.A.H.); Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation [DFG-DU-1872/4-1 to D.D.]; Netherlands Ministry of Education, Culture and Science (OCW); Netherlands Organization for Scientific Research (NWO) [024.003.019 to D.D.]. Funding for open access charge: National Institutes of Health. Conflict of interest statement. None declared.

Publisher Copyright:
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

PubMed: MeSH publication types

Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

UN SDGs

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

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10.1093/nar/gkac1177

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Chinthapatla, R., Sotoudegan, M., Srivastava, P., Anderson, T. K., Moustafa, I. M., Passow, K. T., Kennelly, S. A., Moorthy, R., Dulin, D., Feng, J. Y., Harki, D. A., Kirchdoerfer, R. N., Cameron, C. E., & Arnold, J. J. (2023). Interfering with nucleotide excision by the coronavirus 3^'-to-5^' exoribonuclease. Nucleic acids research, 51(1), 315-336. https://doi.org/10.1093/nar/gkac1177

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abstract = "Some of the most efficacious antiviral therapeutics are ribonucleos(t)ide analogs. The presence of a 3'-to-5' proofreading exoribonuclease (ExoN) in coronaviruses diminishes the potency of many ribonucleotide analogs. The ability to interfere with ExoN activity will create new possibilities for control of SARS-CoV-2 infection. ExoN is formed by a 1:1 complex of nsp14 and nsp10 proteins. We have purified and characterized ExoN using a robust, quantitative system that reveals determinants of specificity and efficiency of hydrolysis. Double-stranded RNA is preferred over single-stranded RNA. Nucleotide excision is distributive, with only one or two nucleotides hydrolyzed in a single binding event. The composition of the terminal basepair modulates excision. A stalled SARS-CoV-2 replicase in complex with either correctly or incorrectly terminated products prevents excision, suggesting that a mispaired end is insufficient to displace the replicase. Finally, we have discovered several modifications to the 3'-RNA terminus that interfere with or block ExoN-catalyzed excision. While a 3'-OH facilitates hydrolysis of a nucleotide with a normal ribose configuration, this substituent is not required for a nucleotide with a planar ribose configuration such as that present in the antiviral nucleotide produced by viperin. Design of ExoN-resistant, antiviral ribonucleotides should be feasible.",

author = "Rukesh Chinthapatla and Mohamad Sotoudegan and Pankaj Srivastava and Anderson, {Thomas K.} and Moustafa, {Ibrahim M.} and Passow, {Kellan T.} and Kennelly, {Samantha A.} and Ramkumar Moorthy and David Dulin and Feng, {Joy Y.} and Harki, {Daniel A.} and Kirchdoerfer, {Robert N.} and Cameron, {Craig E.} and Arnold, {Jamie J.}",

note = "Funding Information: Our thanks to Roel Fleuren and Efra Rivera-Serrano for their contributions to the production of figures and models. We thank Stewart Shuman for providing the expression plasmid for RtcA. National Institutes of Health [R01AI161841 to C.E.C., J.J.A., D.D.; P01CA234228, R01GM110129 to D.A.H., R01AI158463 to R.K.]; University of Minnesota, Office of the Vice President for Research, Grant-in-Aid (to D.A.H.); Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg, the German Research Foundation [DFG-DU-1872/4-1 to D.D.]; Netherlands Ministry of Education, Culture and Science (OCW); Netherlands Organization for Scientific Research (NWO) [024.003.019 to D.D.]. Funding for open access charge: National Institutes of Health. Conflict of interest statement. None declared. Publisher Copyright: {\textcopyright} The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.",

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AU - Sotoudegan, Mohamad

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AU - Anderson, Thomas K.

AU - Moustafa, Ibrahim M.

AU - Passow, Kellan T.

AU - Kennelly, Samantha A.

AU - Moorthy, Ramkumar

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