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

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 possibilitie...

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Published in:Nucleic acids research 2023-01, Vol.51 (1), p.315-336
Main Authors: Chinthapatla, Rukesh, Sotoudegan, Mohamad, Srivastava, Pankaj, Anderson, Thomas K, Moustafa, Ibrahim M, Passow, Kellan T, Kennelly, Samantha A, Moorthy, Ramkumar, Dulin, David, Feng, Joy Y, Harki, Daniel A, Kirchdoerfer, Robert N, Cameron, Craig E, Arnold, Jamie J
Format: Article
Language:English
Subjects:
Antiviral Agents - pharmacology
COVID-19 Drug Treatment
Drug Design
Exoribonucleases - metabolism
Humans
Nucleic Acid Enzymes
Ribonucleotides - chemistry
RNA, Viral - genetics
RNA, Viral - metabolism
SARS-CoV-2 - genetics
SARS-CoV-2 - metabolism
Viral Nonstructural Proteins - metabolism
Virus Replication - genetics
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkac1177