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.
Bibliographical noteFunding 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.
© 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