An Evolutionarily Conserved Strategy for Ribosome Binding and Host Translation Inhibition by β-coronavirus Non-structural Protein 1

[Display omitted] •α- and β-CoV Nsp1s inhibit gene expression.•The CTD of all β-CoV Nsp1s confers high-affinity ribosome binding despite low sequence conservation.•Conservation in surface charge, rather than amino acid sequence, mediates ribosome binding of beta-CoV Nsp1s.•The Nsp1 CTD is an inefficient translation inhibitor that functions by recruiting the Nsp1 NTD to the ribosome.•A cis-acting viral RNA element has co-evolved to fine-tune SARS-CoV-2 Nsp1 function, but does not provide similar protection against Nsp1 from related viruses. An important pathogenicity factor of SARS-CoV-2 and related coronaviruses is Non-structural protein 1 (Nsp1), which suppresses host gene expression and stunts antiviral signaling. SARS-CoV-2 Nsp1 binds the ribosome to inhibit translation through mRNA displacement and induces degradation of host mRNAs. Here we show that Nsp1-dependent host shutoff is conserved in diverse coronaviruses, but only Nsp1 from β-Coronaviruses (β-CoV) inhibits translation through ribosome binding. The C-terminal domain (CTD) of all β-CoV Nsp1s confers high-affinity ribosome binding despite low sequence conservation. Modeling of interactions of four Nsp1s with the ribosome identified the few absolutely conserved amino acids that, together with an overall conservation in surface charge, form the β-CoV Nsp1 ribosome-binding domain. Contrary to previous models, the Nsp1 ribosome-binding domain is an inefficient translation inhibitor. Instead, the Nsp1-CTD likely functions by recruiting Nsp1′s N-terminal “effector” domain. Finally, we show that a cis-acting viral RNA element has co-evolved to fine-tune SARS-CoV-2 Nsp1 function, but does not provide similar protection against Nsp1 from related viruses. Together, our work provides new insight into the diversity and conservation of ribosome-dependent host-shutoff functions of Nsp1, knowledge that could aid future efforts in pharmacological targeting of Nsp1 from SARS-CoV-2 and related human-pathogenic β-CoVs. Our study also exemplifies how comparing highly divergent Nsp1 variants can help to dissect the different modalities of this multi-functional viral protein.