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

Guillen, J. V.; Tokamov, S. A.; Glaunsinger, B. A.

doi:10.64898/2026.09.22.753304

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bioRxiv · 10.64898/2026.09.22.753304

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

Guillen, J. V.·Tokamov, S. A.·Glaunsinger, B. A.

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Abstract

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 beta-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.

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Guillen, J. V., Tokamov, S. A., Glaunsinger, B. A.. 2026-09-23. Mutational screening reveals a cluster of residues within the SARS-CoV-2 nsp1 N-terminus that confers RNA-targeting selectivity. https://doi.org/10.64898/2026.09.22.753304

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