In vitro reconstitution of SARS CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g

Figures

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  Figure 1.. SARS CoV-2 Nsp1-induced cleavage of mRNA during 5’end-dependent initiation. (A) Schematic representation of (CAA)4-MF β-globin and native β-globin mRNAs, annotated to show the initiation codon (bold) and sites of Nsp1-mediated cleavage (red arrows). (B-D, F-H) Cleavage of (B-D, F, G) (CAA) 4-MF β-globin and (H) native β-globin mRNAs depending on the presence of (B-E, G, H) wt, (G) RK124–125AA or KH164–165AA SARS CoV-2 Nsp1 and translational components as indicated, assayed by toe-printing, or where indicated, by primer extension by reverse transcriptase after phenol extraction of mRNA. (E) The influence of wt SARS CoV-2 Nsp1 on elongation by 80S initiation complexes pre-assembled on (CAA) 4-MF β-globin mRNA, assayed by toe-printing. (B-H) Positions of the full-length (f.l.) cDNA, ribosomal complexes, and cleavage sites (red arrows) are shown on the sides of the panels. Lanes C, T, A, and G show the corresponding sequence derived using the same primer as used for primer extension.

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  Figure 2.. SARS CoV-2 Nsp1-induced cleavage of mRNA during EMCV IRES-mediated initiation. (A) Schematic representation of the EMCV IRES and the proximal coding region, annotated to show IRES domains H-L, the location of inactivating CAG529–531GUC substitutions, the initiation codon (bold) and sites of Nsp1-mediated cleavage (red arrows). (B-F) Cleavage of (B-F) wt and (D) CAG529–531GUC mutant EMCV IRES mRNA depending on the presence of (B-F) wt SARS CoV-2 Nsp1, (C) wt SARS CoV Nsp1 or (F) RK124–125AA SARS CoV-2 Nsp1 and translational components as indicated, assayed by toe-printing, or where indicated, by primer extension by reverse transcriptase after phenol extraction of mRNA. Positions of the full-length (f.l.) cDNA, ribosomal complexes, and cleavage sites (red arrows) are shown on the sides of the panels. Lanes C, T, A, and G show the corresponding sequence derived using the same primer as used for primer extension. Separation of lanes in panels C and D by white lines indicates that these parts were juxtaposed from the same gel.

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  Figure 3.. SARS CoV-2 Nsp1-induced cleavage of mRNA during CrPV and HCV IRES-mediated initiation. (A) Schematic representation of the CrPV IRES with the proximal cognate coding region annotated to show IRES domains 1–3, pseudoknots PKI, PKII and PKIII, stem-loops SL2.1 and SL2.3, inactivating substitutions in domain 2, the P-site codon, the positions of toeprints of IRES-bound 40S subunits and sites of Nsp1-mediated cleavage (red arrows). (B) The influence of wt and KH164–165AA SARS CoV-2 Nsp1 on association of CrPV IRES mRNA with 40S subunits and 80S ribosomes assayed by toe-printing. (C-E) Cleavage of (C-E) wt CrPV IRES mRNA or (E) inactivated SL2.3 and PKIII mutant CrPV IRES mRNA, depending on the presence of wt SARS CoV-2 Nsp1 and translational components as indicated, assayed by toe-printing, or where indicated, by primer extension by reverse transcriptase after phenol extraction of mRNA. Positions of the full-length (f.l.) cDNA, 40S/IRES ribosomal complexes, and cleavage sites (red arrows) are shown on the sides of the panels. (F) Cleavage of wt EMCV IRES mRNA depending on the presence of wt SARS Cov-2 Nsp1 and translational components as indicated, assayed by primer extension by…

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  Figure 4.. Determination of eIF3g regions required for Nsp1-induced cleavage of CrPV IRES mRNA. (A) Left panel - model of the Alphafold structure of human eIF3g labeled to show the RRM domain and amino acid residues at the borders of eIF3g truncation mutants (black arrows); right panel - recombinant purified eIF3g truncation mutants, assayed by SDS-PAGE followed by SimplyBlue staining. (B) The activities of eIF3g truncation mutants (panel A) in supporting cleavage of wt CrPV IRES mRNA induced by wt SARS CoV-2 Nsp1, assayed by toe-printing. Positions of the full-length (f.l.) cDNA, 40S/IRES ribosomal complexes, and cleavage sites (red arrows) are shown on the left. (C) Amino acid sequence alignment of the RRM domain of human eIF3g with that of other species, done using Clustal Omega (Sievers et al., 2011). The figure was generated using ESPript 3.0 (Robert and Gouet, 2014). Residues boxed in red are identical. Secondary structure elements from the structure of the human eIF3g RRM domain (PDB: 2CQ0) are represented schematically above and the RNP1 and RNP2 motifs are indicated below the sequence alignment. The amino acid sequence of H. sapiens eIF3g (NP_003746.2) between residues 233 and 320…

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  Figure 5.. The importance of eIF3g for Nsp1-induced cleavage of β-globin and EMCV IRES-containing mRNAs. (A) Purified native eIF3 from HeLa cells and eIF3 containing FLAG-tagged wt or mutant eIF3g expressed in Expi293 cells, analyzed by SDS-PAGE followed by SimplyBlue staining. The positions of individual subunits of eIF3 are indicated. (B-C) The activity of eIF3 containing FLAG-tagged wt or mutant eIF3g in 48S complex formation on (B) (CAA)4-MF β-globin and (C) EMCV IRES mRNAs in the presence of indicated translational components with/without Nsp1, assayed by toe-printing. (D-E) Nsp1-induced cleavage of (CAA)4-MF β-globin, CrPV IRES and EMCV IRES mRNAs depending on eIF3 containing either wt or mutant FLAG-tagged eIF3g, assayed by primer extension by reverse transcriptase after phenol extraction of mRNA. (B-E) Positions of the full-length (f.l.) cDNA, 48S complexes and cleavage sites (red arrows) are shown on the sides of the panels.

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  Figure 6.. Mutational analysis of SARS CoV-2 Nsp1 (A-E) Cleavage of (A-C) EMCV IRES, (D) CrPV IRES or (E) (CAA)4-MF β-globin mRNAs in the presence of wt and Ala substitution mutant SARS CoV-2 Nsp1 and indicated translational components, assayed by toe-printing, or where indicated, by primer extension by reverse transcriptase after phenol extraction of mRNA. Mutations having the strongest effect on Nsp1-induced cleavage in panels A-C are in red, mutations causing a moderate effect are in orange, and mutations that don’t affect cleavage are in black. Positions of the full-length (f.l.) cDNA and cleavage sites (red arrows) are shown on the sides of the panels. Lanes C, T, A, and G show the corresponding sequence derived using the same primer as used for primer extension. (F-H) Inhibition of 48S complex formation on the EMCV IRES by Nsp1 Ala substitution mutants with the lowest cleavage-inducing activity, assayed by toe-printing. Positions of the full-length (f.l.) cDNA and 48S complexes are shown on the sides of the panels. Lanes C, T, A, and G show the corresponding sequence derived using the same primer as used for primer extension. Separation of lanes in panels D, G and H by white lines…

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  Figure 7.. Chemical inhibition of SARS CoV-2 Nsp1-induced cleavage of mRNA (A, C-F) The influence of FDA-approved drugs on Nsp1-induced mRNA cleavage and inhibition of 48S complex formation on (A, C, F) CrPV IRES, (D) HCV IRES and (E) (CAA)4-MF β-globin mRNAs assayed by toe-printing. Positions corresponding to the full-length (f.l.) cDNA, 40S/IRES and eIF3/IRES contacts, 48S complexes and cleavage sites (red arrows) are shown on the sides of the panels. (B) Structures of Mitoxantrone, Ametantrone and Pixantrone.