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

• Published in: Nucleic acids research Vol. 51; no. 1; pp. 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: Journal Article
• Language: English
• Published: England Oxford University Press 11.01.2023
• 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
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkac1177

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.