Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40S ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40S and various native Nsp1-40S and -80S complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.

Fig. 1. Nsp1 interacts with 40S ribosomal subunits and inhibits translation.

(A) Domain organization of Nsp1 and sequence alignment of the C-terminal segment (red line) of Nsp1 from seven human CoVs. The KH motif is marked. (B) In vitro binding assay of GST-TEV (GST)–tagged Nsp1 and Nsp1-mt from SARS-CoV-1 (SCoV-1) and SCoV-2 with human 40S and 60S ribosomal subunits. A Coomassie-stained SDS–polyacrylamide gel electrophoresis (PAGE) gel for inputs and eluates is shown. GST, glutathione S-transferase; MW, molecular weight markers. (C) Polyribosome gradient analysis of HEK293T lysate (control) and lysate from HEK293T cells transiently transfected with 3×FLAG-tagged Nsp1 and Nsp1-mt constructs from SCoV-1 and SCoV-2 and Western blot analysis (anti-FLAG antibody; separate blots). (D) Western blot (top, anti-V5 antibody) and SDS-PAGE analysis (bottom) of cell-free in vitro translation of a capped reporter mRNA with rabbit reticulocytes (RRL) and HeLa S3 lysate. Controls 1 and 2, with and without capped reporter mRNA, respectively. A Coomassie-stained SDS-PAGE gel of the applied (His)₆–tobacco etch virus (His₆)–tagged Nsp1 constructs is shown below. (E) Quantification of luciferase activity in HEK293T cells transfected with indicated 3×FLAG-tagged proteins and in vitro–transcribed firefly luciferase mRNA. Bars represent means ± SEM (n = 6 samples). RLU, relative light units. Representative immunoblots of whole-cell lysates (WCL) stained with anti-FLAG and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH). **P < 0.001 [unpaired Student’s t test (Welch correction)].

Fig. 2. Cryo-EM structures of Nsp1-bound ribosomal complexes.

(A) SDS-PAGE analysis of reconstituted Nsp1-40S complexes. Nsp1 is labeled with an asterisk. MW, molecular weight markers. (B) Reconstituted Nsp1-40S structure with Nsp1 shown in pink; rRNA and proteins are shown in yellow. Additional density between uS3 and h16 assigned to the N-terminal fold of Nsp1 is shown. bk, beak; pf, platform; lf, left foot; rf, right foot. (C) C-terminal helix 1 and 2 of Nsp1 with corresponding densities. (D) Cross-section of the 40S, highlighting the central position of Nsp1 within the mRNA tunnel. The putative position of the N-terminal domain of Nsp1 is schematically indicated [models are based on PDB-2HSX (21) and PDB-6Y0G (47)]. (E) SDS-PAGE analysis of Nsp1-ribosomal complexes affinity purified from HEK293T cells. Proteins identified in the cryo-EM structures were labeled according to mass spectrometry analysis (data S1). (F to N) Cryo-EM maps of affinity-purified Nsp1-ribosomal complexes. Additional factors are colored and labeled accordingly.

Fig. 3. Molecular basis of Nsp1 ribosome interaction and inhibition.

(A) Cryo-EM map of in vitro–reconstituted Nsp1-40S and segmented density of Nsp1-C, uS3 (residues 97 to 153 and 168 to 189), uS5 (102 to 164), and rRNA helix h18 with the corresponding models. Interacting residues are shown as sticks. (B) Nsp1-C surface, colored by electrostatic potential from −5 (red) to +5 (blue). (C) Model of Nsp1-C and surface representation of the models of uS3 (residues 97 to 153 and 168 to 189), uS5 (102 to 164), and rRNA helix h18. Molecular interactions between Nsp1 and the ribosome are shown. (D) mRNA entry channel; 40S head is removed. Nsp1-C occupies the mRNA path [PDB-6Y0G (47)]. (E) K164 and H165 of Nsp1 bind to a pocket on h18. (F) R171 and R175 of Nsp1 bind to the phosphate backbone of h18. (G) Negatively charged residues D152, E155, and E159 of α1 interact with uS3. (H) The hydrophobic interface of α1 and α2 binds to a hydrophobic patch on uS5. (I) Schematic summary of the interaction of Nsp1-C with uS3, uS5, and h18; residues belonging to α1 and α2 are colored pink. A, adenine; C, cytosine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, guanine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; R, arginine; T, threonine; U, uracil; V, valine; W, tryptophan; Y, tyrosine.

Fig. 4. Inhibition of the innate immune response by SARS-CoV-2 Nsp1.

(A) Quantification of IFN-β promoter–controlled firefly luciferase activity in HEK293T cells transiently expressing 3×FLAG-tagged or nontagged (RV P) proteins. Cells were infected with SeV or left uninfected. Representative immunoblots of whole-cell lysates (WCLs) stained with anti-RV P, anti-FLAG, and anti-GAPDH are shown (bottom panel). (B) Enzyme-linked immunosorbent assay results for IFN-β, IFN-λ1, and IL-8 in the supernatant of HEK293T cells transiently expressing 3×FLAG-tagged proteins and infected with SeV (top panel) for 24 hours. Quantitative polymerase chain reaction (qPCR) results for corresponding mRNAs are shown in the bottom panel. (C and D) Quantification of ISRE promoter– controlled firefly luciferase activity in HEK293T cells transiently expressing 3×FLAG-tagged proteins in single amounts (C) or increasing amounts (D) and treated with 1000 U/ml IFN-β as indicated. Representative immunoblots of WCLs stained with anti-FLAG and anti-GAPDH are shown in the bottom panels. (E) Representative immunoblots and quantification of WCLs of HEK293T cells stimulated with 200 U/ml IFN-β and stained for endogenous RIG-I, ISG15, and GAPDH. qPCR results for the corresponding mRNAs are shown in the bottom two panels. In (A), (C), and (D), bars represent means ± SEM of six samples; in (B) and (E), bars represent means ± SEM of three samples. ns, not significant; *P < 0.01; ***P < 0.0001 [unpaired Student’s t test (Welch correction)].