. Collectively, our results solidify the multiple critical roles of nsp14-ExoN across CoV genera and establish new approaches for rescuing and analyzing loss-of-function substitutions in studies of CoV replication, pathogenesis, and evolution. IMPORTANCE: Coronaviruses (CoV) are important human pathogens causing hundreds of millions of infections and millions of deaths over the past 20 years. The study of how these viruses multiply and cause disease identifies points of attack for therapeutics. Using a high-throughput genetic approach, we systematically inactivated an essential enzyme CoV needs for replication called ExoN. We show that without ExoN, CoV replication fidelity and fitness are reduced in cell culture. Replication without ExoN in mice was diminished but could be partially restored in mice that lack key components of the immune response. Altogether, we reveal new insights into the complexities of CoV replication and virus and host interactions, which could be leveraged for the development of novel multifaceted therapeutics that attack the ever-expanding functions of the CoV replication complex in replication and pathogenesis.
Anderson-Daniels et al. (Wed,) studied this question.