Fig. 3: SARS-CoV-2 Nsp1 inhibits translation in HeLa cell lysates by binding to the 40S ribosomal subunit.
From: SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation
a, The C-terminal domain of Nsp1 binds to the mRNA entry site of 40S (red), while the N-terminal domain (gray) is flexibly disposed. Mutants in helix 2 (Y154A/F157A in green), in the short connecting loop (K164A/H165A in orange), in helix 3 (R171E/R175E in blue) and a truncation of the C-terminal domain were generated. In the Trx1-Linker-Cter Nsp1 construct, the N-terminal domain was replaced by Escherichia coli Trx1 (purple). b, Relative RLuc activity measurements of in vitro translation reactions normalized to the reaction in the absence of Nsp1 (0 μM). Data are presented as mean values ± standard deviations of three biological replicates (sets of translation reactions) averaged after three measurements; mean values of each biological replicate are indicated by dots. One-way analysis of variance (ANOVA) analysis is compared to the 0 μM Nsp1 condition. NS, not statistically significant (>0.05); **P < 0.01, ****P < 0.0001. c, In the in vitro binding assay, Nsp1 mutants were added to 40S and 60S ribosomal subunits and loaded on a 30% (wt/vol) sucrose cushion. SN, supernatant; P, pellet. d, Scheme of the in vitro synthesized capped (black dot) and polyadenylated reporter mRNAs coding for Renilla Luciferase (hRLuc). e, RLuc activity measurements of in vitro translation reactions normalized to the readout of RLuc reporter mRNA. Bars, mean values and dots are defined in b. P value of an unpaired t-test, 0.014. f, Titration of WT Nsp1 against RLuc and FL-RLuc reporter mRNAs. Relative RLuc activities were normalized to the untreated sample (0 μM). Bars, mean values and dots are as defined in b. One-way ANOVA analysis is compared to the 0 μM Nsp1 condition and the P values were all <0.0001. ORF, open reading frame. Source data for b, e and f and uncropped gel images for c are available online.
