Loss of nsp14-exonuclease activity impairs the replication, proofreading, fitness and pathogenesis of SARS-CoV-2
Coronaviruses (CoVs) and other members of the order Nidovirales replicate their ~30kb positive sense RNA genomes with higher fidelity than other RNA viruses, mediated by the proofreading and recombination activities of the exoribonuclease domain of replicase nonstructural protein 14 (nsp14-ExoN). We previously reported that loss-of-function amino acid substitutions in nsp14-ExoN (ExoN-) in Betacoronaviruses (Beta-CoVs) murine hepatitis virus (MHV) and SARS-CoV (D90A and E92A) are viable but significantly impaired in replication, recombination, fitness, fidelity, virulence and immune antagonism. However, identical substitutions in MERS-CoV and SARS-CoV-2 have been proposed to be lethal for virus replication. Here, we report a saturation mutagenesis approach facilitating the recovery and analysis of several constellations of SARS-CoV-2 nsp14 ExoN-inactivating, loss-of-function substitutions, including the canonical D90A and E92A. Biochemical assays with purified WT or ExoN- nsp10-14 fusion proteins confirmed that active site substitutions abolished ExoN activity (ExoN-). SARS-CoV-2 ExoN- viruses had impaired replication, RNA synthesis, and recombination, as well as decreased replication fidelity and loss of fitness in vitro. ExoN- viruses were significantly attenuated for replication in human primary airway epithelial cells and were attenuated for replication and pathogenesis in WT mice as well as the highly susceptible K18 transgenic mice. In the absence of interferon signaling in vivo, SARS-CoV and SARS-CoV-2 ExoN- viral replication could be partially restored. These results demonstrate that SARS-CoV-2 ExoN- are viable but highly impaired for replication, fitness, and fidelity in vitro as well as innate immune antagonism and pathogenesis in vivo. Collectively, our results solidify the multiple critical roles for nsp14-ExoN across CoV genera and establish new approaches for rescue and analysis of loss-of-function substitutions for studies of CoV replication, pathogenesis, and evolution.