Clinically observed deletions in SARS‐CoV‐2 Nsp1 affect its stability and ability to inhibit translation

Fig. 1. Nsp1 variants with amino acid deletions located outside of the mRNA tunnel‐targeting CTD differ in their ability to inhibit translation. (A) Structural organization of SARS‐CoV‐2 Nsp1 protein showing the N‐terminal and C‐terminal domains (NTD and CTD) as well as the regions containing naturally occurring deletions [25, 26]. Also denoted are amino acids R124‐K125 necessary for host mRNA cleavage [11] and the R164‐K165 pair reported to be necessary for the interaction with the 40S mRNA entry site [12]. The secondary structure elements of wild‐type Nsp1 are shown above the domain organization. (B) Schematic of the viral mRNA (mRNA reporter construct containing the 5′ UTR of SARS‐CoV‐2) and the nonviral mRNA (mRNA reporter construct with 5ʹ non‐SARS‐CoV‐2 UTR) followed by a firefly luciferase open reading frame, with 5′ cap and 3′ poly A tail. (C) Results of the in vitro translation assay to assess the translation efficiency of the reporter mRNA. Graph showing luminescence signal in response to firefly luciferase activity in HEK293F translational lysate at the end of the reaction, in the absence and presence of increasing concentrations (0.1, 0.5, 1.0, 2.0, and 3.0 μm) of recombinant…

Fig. 2. Deletion of residues 79–89 does not abrogate Nsp1 binding to 40S ribosomal subunit. HEK293F translation lysate was combined with His6‐tagged Nsp1 protein (wt or Δ79–89) and resolved by ultracentrifugation on a 10–50% sucrose gradient. rRNA and His6‐Nsp1 protein in individual fractions were visualized using agarose electrophoresis and western blotting, respectively.

Fig. 3. The ∆79–89 deletion variant of SARS‐CoV2 Nsp1 has a drastically reduced thermal stability. (A) Raw data of thermal shift assays [34] with Nsp1 variants using SYPRO Orange. (B) Raw TSA data of the deletion variant ∆79–89. (C) Nonlinear regression curve fit (Boltzmann sigmoidal) of the unfolding curves of Nsp1 variants (except that of the ∆79–89 protein) to calculate the melting temperature. (D) Same as (C), for the ∆79–89 variant. (E) Tabular presentation of the result of the nonlinear fitting of the unfolding curve of all the proteins. Statistical significance of T m change from wt was estimated for each deletion variants by unpaired t test and is indicated in the table. P value for ∆85 = 0.002, for ∆82–83, 85 = 0.066, for ∆79–89 = 0.002, and for ∆141–143 = 0.423. n.s., not significant. **P < 0.01.

Fig. 4. Predicted β‐barrel integrity correlates with altered thermal stability of Nsp1 deletion variants. (A) Cartoon representation of the experimentally determined structure of the Nsp1 N‐terminal β‐barrel domain (PDB ID: 7K7P, [27]). (B) Cartoon representation of the predicted wt Nsp1 structure (gray), truncated to the same boundaries as for (A). In red highlighted are residues 79–89. (C) Cartoon representation of the predicted Δ79–89 Nsp1 structure (orange), truncated to the same boundaries as for (A). In red highlighted is the disordered region, which in (B) forms the β‐strands β3–β5. (D) Topological drawing of the wild‐type Nsp1 fold with α‐helices shown as black tubes and the β‐strands as arrows. β‐strands forming the β‐barrel are shown in gray and non‐β‐barrel forming strands in light gray. Highlighted in red is the β‐strand part deleted in Δ79–89 Nsp1. (E) Topological drawing of the Δ79–89 Nsp1 fold. α‐helices are shown as black tubes and the β‐strands as orange arrows. Highlighted in red is the disordered region as in (C). (D, E) Dashed‐lined box highlights the location of change between the predicted wt and Δ79–89 folds.