Abstract
Coronaviruses (CoVs) maintain large RNA genomes that frequently undergo mutations and recombination, contributing to their evolution and emergence. In this study, we find that SARS-CoV-2 has greater RNA recombination frequency than other human CoVs. In addition, CoV RNA recombination primarily occurs at uridine (U)-enriched RNA sequences. Therefore, we next evaluate the role of SARS-CoV-2 NSP15, a viral endonuclease that targets uridines (EndoU), in RNA recombination and virus infection. Using a catalytically inactivated EndoU mutant (NSP15(H234A)), we observe attenuated viral replication in vitro and in vivo. However, the loss of EndoU activity also dysregulates inflammation resulting in similar disease in vivo despite reduced viral loads. Next-generation sequencing (NGS) demonstrates that loss of EndoU activity disrupts SARS-CoV-2 RNA recombination by reducing viral sub-genomic mRNA but increasing recombination events that contribute to defective viral genomes (DVGs). Overall, the study demonstrates that NSP15 plays a critical role in regulating RNA recombination and SARS-CoV-2 pathogenesis.