Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme

Liu, Chang; Shi, Wei; Becker, Scott T; Schatz, David G; Liu, Bin; Yang, Yang

Coronavirus 3'-5' exoribonuclease (ExoN), residing in the nonstructural protein (nsp) 10-nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the virus life cycle. ExoN also recognizes and excises nucleotide analog inhibitors incorporated into the nascent RNA, undermining the effectiveness of nucleotide analog-based antivirals. Here, we present cryo-electron microscopy structures of both wild-type and mutant SARS-CoV-2 nsp10-nsp14 in complex with an RNA substrate bearing a 3'-end mismatch at resolutions ranging from 2.5 Ã… to 3.9 Ã…. The structures reveal the molecular determinants of ExoN substrate specificity and give insight into the molecular mechanisms of mismatch correction during coronavirus RNA synthesis. Our findings provide guidance for rational design of improved anti-coronavirus therapies.