SARS-CoV-2 nsp1 mediates broad inhibition of translation in mammals

Abstract

SARS-CoV-2 nonstructural protein 1 (nsp1) promotes innate immune evasion by inhibiting host translation in human cells. However, the role of nsp1 in other host species remains elusive, especially in bats which are natural reservoirs of sarbecoviruses and possess a markedly different innate immune system than humans. Here, we reveal that SARS-CoV-2 nsp1 potently inhibits translation in bat cells from Rhinolophus lepidus, belonging to the same genus as known sarbecovirus reservoirs hosts. We determined a cryo-electron microscopy structure of SARS-CoV-2 nsp1 bound to the Rhinolophus lepidus 40S ribosome and show that it blocks the mRNA entry channel via targeting a highly conserved site among mammals. Accordingly, we found that nsp1 blocked protein translation in mammalian cell lines from several species, underscoring its broadly inhibitory activity and conserved role in numerous SARS-CoV-2 hosts. Our findings illuminate the arms race between coronaviruses and mammalian host immunity (including bats), providing a foundation for understanding the determinants of viral maintenance in bat hosts and spillovers.

Fig 1.. SARS-CoV-2 nsp1 suppresses translation in human (HEK293T) and Rhileki (bat) cells.

(A) mRNA construct design and schematic of the fluorescence-based translation assay. 200 ng of eGFP-encoding mRNA was transfected with 0.01-1 ng of a wildtype or mutant nsp1 mRNA to enable visualization and quantification of translation inhibition. mRNAs are all 5’ capped (shown by the triangular grey shapes) and have poly(A) tails at the 3’ end. SC2: SARS-CoV-2. 5’-UTR: 5’ untranslated region. 3xFLAG: triple flag tag. nsp1 KH mutant: nsp1 K164A/H165A double mutant. (B) Live-cell fluorescence imaging of GFP expression in the presence (or absence) of varying amounts of wildtype nsp1 mRNA or of the K164A/H165A double mutant (mut) mRNA using Rhileki and HEK293T cells. Scale bar: 100 μm. See Fig S1 for GFP expression levels. (C) Quantification of the nsp1-mediated dose-dependent inhibition of translation based on normalized GFP fluorescence intensity across the entire field of view. Bars represent the mean of six biological replicates shown as individual data points with error bars showing standard deviation. Each biological replicate is a mean of six technical replicates. Transfections with wildtype and mutant nsp1 mRNAs were compared to the GFP-only control using one-way ANOVA and follow-up Dunnet’s T3 multiple comparisons tests. P-values reported are adjusted for multiplicity. **: p<0.01, ***: p<0.005.

Figure 2.. Architecture of the Rhileki bat 40S ribosome bound to SARS-CoV-2 nsp1.

(A) Comparison of the 40S Rhileki ribosome and the human ribosome organization (PDB: 8PPK). Each ribosomal protein is colored distinctly and labeled whereas the 18S rRNA is rendered as a purple surface. The box showing the nsp1 binding interface is zoomed-in in panel B. (B) Closeup view of SARS-CoV-2 nsp1 (pink) within the mRNA entry channel of the bat 40S ribosome and its interacting partners uS5 (blue), uS3 (light green), eS30 (yellow), and helix 18 (h18) of the 18S rRNA (purple). (C) Key interactions of nsp1 CTD with rRNA, uS5, and uS3. Polar interactions are shown as black dotted lines. Select residues are rendered with semi-transparent density. (D) Interaction map of the SARS-CoV-2 nsp1 CTD with the 40S ribosome. The CTD residues are connected to key ribosomal interactors through dotted lines.

Figure 3.. SARS-CoV-2 nsp1 inhibits translation in a wide range of mammalian species.

(A) Live-cell fluorescence imaging of GFP expression in the presence (or absence) of varying amounts of wildtype nsp1 or of the K164A/H165A nsp1 mutant (mut) mRNA in Crandell-Rees feline kidney epithelial cells (CRFK), canine fibroblasts cells (A72), American mink epithelial cells (Mv 1 Lu), Southern red muntjac deer fibroblast cells (Muntjac), and pig testis fibroblast cells (ST). Scale bar: 100 μm. (B) Quantification of the nsp1-mediated dose-dependent inhibition of translation based on normalized GFP fluorescence intensity using the entire field of view. Bars represent the mean of six biological replicates shown as individual points. Each biological replicate is a mean of six technical replicates. Transfection with wildtype and mutant nsp1 mRNAs were compared to the GFP-only control using one-way ANOVA and follow-up Dunnet’s T3 multiple comparisons tests. See Fig S1 for GFP expression levels. P-values reported are adjusted for multiplicity. **: p<0.01, ***: p<0.005, ****: p<0.001.