Nsp1 from divergent coronaviruses has conserved endonuclease activity

All alpha- and beta-coronaviruses encode nonstructural protein 1 (Nsp1), a major virulence factor that restricts host gene expression. Herein, using Nsp1 from divergent alpha- and beta-coronaviruses (SARS-CoV-2, MERS-CoV, and HCoV-229E), we reveal all tested coronavirus Nsp1 proteins have intrinsic endonuclease activity. Furthermore, this endonuclease function is abolished when a conserved arginine-lysine motif in the N-terminal domain (NTD) is disrupted. For SARS-CoV-2 Nsp1, the eukaryotic Initiation Factor 3g (eIF3g) and the 40S ribosome act as cofactors for enhancing the endonuclease function, but these host factors are not conserved for the endonuclease function of MERS-CoV and HCoV-229E Nsp1. We propose that SARS-CoV-2 Nsp1 uses eIF3g and the 40S ribosome to enhance its affinity for RNA and target host mRNAs. Similar enhancement in endonuclease activity is observed when Nsp1 from SARS-CoV-2, MERS-CoV, and HCoV-229E are cis-tethered to an RNA-binding module. Collectively, our results show that endonuclease activity is intrinsic to Nsp1 across divergent coronavirus genera, and this endonuclease activity is likely targeted towards host mRNAs via a diverse set of host mRNA binding cofactors.