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 5′ UTR, coding region, stop codon, sites of Nsp1-mediated cleavage (red arrows), and the position of the toeprinting primer. (B–D) The influence of wt Nsp1 on 48S complex formation on (CAA)4-MF mRNA and on its integrity in the presence of the indicated translation components, assayed by toeprinting or, where indicated, by primer extension by RT after phenol extraction of mRNA. (E) Nsp1-induced cleavage of 5′[32P]cap-labeled (CAA)4-MF mRNA depending on the presence of the indicated translation components, assayed directly after phenol extraction. A 10-nt-long 5′[32P]-labeled oligo RNA was used as a marker. (F) Time course of Nsp1-induced cleavage of (CAA)4-MF β-globin in the presence of the indicated translation components, assayed by primer extension by RT after phenol extraction of mRNA. (G) The influence of wt Nsp1 on (CAA)4-MF β-globin mRNA in preassembled 80S initiation complexes and their ability to undergo elongation, assayed by toeprinting. (H) The influence of wt and RK124–125AA or KH164–165AA…

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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, the inactivating CAG529–531GUC substitutions, the initiation codon, the position of the toeprinting primer, and sites of Nsp1-induced cleavage (red arrows). (B–F) The influence of wt SARS-CoV-2 Nsp1 (B), wt SARS-CoV-2 and wt SARS-CoV Nsp1s (C), wt SARS-CoV-2 Nsp1 (D,E), and RK124–125AA SARS-CoV-2 Nsp1 mutant (F) on 48S complex formation on wt (B,C,E,F) and CAG529–531GUC mutant (D) EMCV IRES mRNAs as well as on mRNA integrity in the presence of the indicated translation components, assayed by toeprinting or, where indicated, by primer extension by RT after phenol extraction of mRNA. Separation of lanes in 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, pseudoknots, stem–loops SL2.1 and SL2.3, inactivating substitutions in domain 2, the P-site codon, the position of 40S/IRES toeprints, the position of the toeprinting primer, and sites of Nsp1-mediated cleavage (red arrows). (B) The influence of wt and KH164–165AA Nsp1 on association of CrPV IRES mRNA with 40S subunits and 80S ribosomes assayed by toeprinting. (C–E) Cleavage of wt (C,D) or inactivated SL2.3 and PKIII mutant (E) CrPV IRES mRNAs depending on the presence of wt Nsp1 and various translation components, assayed by toeprinting or, where indicated, by primer extension by RT after phenol extraction of mRNA. (F) Cleavage of wt EMCV IRES mRNA depending on the presence of wt Nsp1 and translation components as indicated, assayed by primer extension after phenol extraction of mRNA. (G,H) The influence of wt Nsp1 on ribosomal binding and the integrity of the HCV IRES depending on the presence of various translation initiation components, assayed by toeprinting or, where…

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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) Purified eIF3g truncation mutants, assayed by SDS-PAGE followed by SimplyBlue staining. (B) The activities of eIF3g mutants in supporting Nsp1-induced cleavage of wt CrPV IRES mRNA, assayed by toeprinting. (C) Amino acid sequence alignment of the RRM domain of human eIF3g with that of other species, done using Clustal Omega. Residues boxed in red are identical. Secondary structure elements from the structure of the human eIF3g RRM domain (PDB: 2CQ0) are represented schematically above the sequence alignment, and the RNP1 and RNP2 motifs are indicated below the sequence alignment. The amino acid sequence of Homo sapiens eIF3g (NP_003746.2) between residues 233 and 320 is aligned with its Exaiptasia diaphana (XP_020896357), Drosophila melanogaster (NP_650887), Apis mellifera (XP_391934), Schizosaccharomyces pombe (NP_595727), and Saccharomyces cerevisiae (NP_010717) homologs. Residues substituted by Ala are…

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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. (B,C) The activity of eIF3 containing FLAG-tagged wt or mutant eIF3g in 48S complex formation on (CAA)4-MF β-globin (B) and EMCV IRES (C) mRNAs in the presence of the indicated translation components with/without wt Nsp1, assayed by toeprinting. (D,E) The activities of eIF3 containing either wt or mutant FLAG-tagged eIF3g in supporting Nsp1-induced cleavage of (CAA)4-MF β-globin, CrPV IRES, and EMCV IRES mRNAs in the presence of the indicated translation components, assayed by primer extension after phenol extraction of mRNA.

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  Figure 6.. Mutational analysis of SARS-CoV-2 Nsp1. (A–C) Cleavage of EMCV IRES mRNA induced by wt and Ala substitution mutant Nsp1s in the presence of the indicated translation components, assayed by primer extension after phenol extraction of mRNA. (D,E) Cleavage of CrPV IRES (D) and (CAA)4-MF β-globin (E) mRNAs in the presence of wt and Ala substitution mutant Nsp1s and the indicated translation components, assayed by toeprinting. (A–E) Mutations having the strongest effect on Nsp1-induced cleavage in A–C are in red, mutations causing a moderate effect are in orange, and mutations that do not affect cleavage are in black. (F–H) Inhibition of 48S complex formation on the EMCV IRES mRNA by Nsp1 Ala substitution mutants with the lowest cleavage-inducing activity, assayed by toeprinting. Separation of lanes in D, G, and H by white lines indicates that these parts were juxtaposed from the same gels. (I) Ribbon diagram (left panel) and surface charge distribution (right panel) of the Nsp1 NTD (PDB: 7K3N) in ribbon (left panel) and space-filling (right panel) representations, annotated to show residues that contribute strongly (red sticks) or moderately (orange sticks) to Nsp1-induced cleavage…

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  Figure 7.. Chemical inhibition of SARS-CoV-2 Nsp1-induced cleavage of mRNA. (A) Structures of mitoxantrone, ametantrone, and pixantrone. (B–G) The influence of FDA-approved drugs on Nsp1-induced mRNA cleavage and inhibition of 48S complex formation on CrPV IRES (B–D,G), HCV IRES (E), and (CAA)4-MF β-globin (F) mRNAs in the presence of the indicated translation components, assayed by toeprinting.