Mutational screening reveals a cluster of residues within the SARS-CoV-2 nsp1 N-terminus that confers RNA-targeting selectivity

The SARS-CoV-2 nsp1 protein is a virulence factor that broadly inhibits cellular gene expression. Although cellular mRNAs are translationally inhibited by nsp1 and subsequently degraded, viral transcripts possess a 5′ leader sequence (CoV2L) that enables them to escape nsp1-mediated repression. Both transcript targeting selectivity and mRNA decay require coordination by the nsp1 N-terminal domain (NTD) through an unknown mechanism. Here, we generated an alanine-scanning library of mutations encompassing all residues in the nsp1 NTD to gain a deeper understanding of how this domain coordinates nsp1 function. We screened this library for the ability to repress mRNA bearing a host- or CoV2L-derived 5′ untranslated region, revealing two predominant clusters of residues required for target selectivity. These largely comprised adjacent surface-exposed β-sheets on the NTD, whose deletion rendered CoV2L-containing mRNA susceptible to repression and prevented nsp1-induced mRNA decay. This work provides residue-level information on the role of the NTD in distinguishing among mRNA targets and further links nsp1 target selectivity to mRNA decay.