Structural Basis and Rational Design of Nucleotide Analogue Inhibitor Evading the SARS-CoV-2 Proofreading Enzyme

Area of Science:

• Virology
• Molecular Biology
• Drug Discovery

Background:

• Coronaviruses (CoVs) utilize an exoribonuclease (nsp14 ExoN) to proofread nascent RNA, excising mismatched nucleotides or nucleotide analogues (NAs).
• Understanding how NAs evade this proofreading mechanism is crucial for developing effective antiviral therapies against CoVs.

Purpose of the Study:

• To investigate the mechanism by which SARS-CoV-2 nsp14 ExoN cleavage is evaded by specific nucleotide analogues (NAs).
• To identify structural features of NAs that confer resistance to nsp14 ExoN activity.
• To guide the rational design of novel anti-CoV NAs.

Main Methods:

• Utilized chemically synthesized RNA incorporating various NAs at the 3' end.
• Assessed the cleavage activity of SARS-CoV-2 nsp14 ExoN on these modified RNAs.
• Determined cryo-electron microscopy structures of nsp10/14 complexes bound to RNA with NAs.
• Performed mutagenesis studies on key residues within nsp14 ExoN.

Main Results:

• Nsp14 ExoN showed significantly reduced activity on RNA containing sofosbuvir monophosphate (SMP) and AT-9010 monophosphate (ATMP) compared to natural nucleotides or other tested NAs.
• Cryo-EM structures and mutagenesis revealed that residues H95, Q145, and F146 are critical for recognizing the base moiety and positioning NAs for cleavage.
• NAs evade cleavage through a combination of a non-interacting base and a chemically modified ribose.

Conclusions:

• The evasion of nsp14 ExoN cleavage by NAs is dependent on specific base-nucleotide interactions and ribose modifications.
• Two novel NAs were designed based on these findings, demonstrating resistance to nsp14 ExoN cleavage.
• These insights are valuable for the rational design of next-generation anti-coronavirus nucleotide analogues.