The coronavirus proofreading exoribonuclease mediates extensive viral recombination

Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs. Author summary Recombination is an essential part of normal coronavirus replication, required for the generation of the sub-genomic mRNAs as well as defective viral genome (DVGs) and is also implicated in novel strain emergence. However, the molecular mechanisms and determinants of RNA recombination in CoVs are unknown. Here, we compare recombination in 3 divergent beta-coronaviruses; murine hepatitis virus (MHV), MERS-CoV, and SARS-CoV-2. We show that they have striking similarities in the populations of RNA produced and in the sequences surrounding recombination junctions. Further, we demonstrate that the coronavirus proofreading exoribonuclease (nsp14-ExoN) is required to maintain the rates and loci of recombination generated during infection. These data suggest that recombination and the coronavirus exoribonuclease are conserved and important determinants of replication that may be targeted for inhibition and attenuation to control the ongoing pandemic of SARS-CoV-2 and prevent future outbreaks of novel coronaviruses.

Automated summary

Recombination is essential for coronavirus diversity and the emergence of new strains. The mechanisms of CoV recombination are not well understood, but studies have shown extensive and diverse recombination products in CoVs during replication. The nsp14-ExoN protein is crucial for native recombination in CoVs, highlighting its potential as a target for inhibitors to control SARS-CoV-2 and future emerging zoonotic CoVs.