Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

Fig. 1.. NSP1 from SARS-CoV-2 inhibited translation. (A) Schematic of the host model mRNA used in HeLa cell-free IVT assays. The nLuc coding sequence of the mRNA was flanked by the 5′ and 3′ UTRs of human GAPDH. Numbers refer to the nucleotide position in NCBI GenBank accession: AF261085. (B) NSP1 dose–response analysis of GAPDH reporter mRNA IVT in HeLa extract treated with either wild-type (WT; n = 3), a predicted ribosome-binding−deficient mutant (KH/AA; n = 2), or an RNA cleavage-deficient mutant (RK/AA; n = 2) NSP1. The mean response ± SEM (symbols, error bars) and curve fits (lines) from nonlinear regression analysis of the data are plotted. WT IC50 = 510 ± 20 nM (95% CI; R2 = 0. 83), and RK/AA IC50 = 420 ± 11 nM (95% CI; R2 = 0.89). (C) Plot of the mean nLuc relative light unit (RLU) signal from cell-free translation of host and viral reporter mRNAs in the absence and presence (400 nM) of wild-type NSP1. Without NSP1 (light gray), mean translational activities (percent RLU) were compared to GAPDH reporter mRNA in the absence of NSP1 (** = P ≤ 0.0006; and n.s. = P ≥ 0.2, one-way ANOVA). GAPDH (n = 6), 5′ UTR−3′ UTR(S) mRNA (n = 3), 5′ UTR−3′ UTR(L) (n = 3), and 5′ LDR reporter mRNAs…

Fig. 2.. NSP1 associated with 40S subunits and most ribosomal preinitiation complexes. (A) Experimental setup. Using a ZMW system, 40S ribosomal subunits biotinylated on RACK1 were tethered to a neutravidin-coated imaging surface within thousands of individual ZMWs (also see SI Appendix, Fig. S3B). Upon start of data acquisition, Cy3-NSP1 (N-terminal ybbR tag) was added, and fluorescence intensities were monitored. (B) Example single-molecule fluorescence trace that depicts association of Cy3-NSP1 with a tethered eIF1–40S subunit complex. Prior to tethering, 40S subunits were incubated with 30-fold molar excess eIF1. During imaging, eIF1 was present at 1 µM. The association time (Δt) was defined as the time elapsed from the addition of Cy3-NSP1 until the burst of Cy3 fluorescence (green), which signified NSP1 association. The lifetime was defined as the duration of the Cy3 fluorescence signal. (C) Plot of the fraction of ZMWs that contained at least one Cy3-NSP1 binding event ≥∼5 s in duration in the indicated conditions at 20 °C. Error bars represent 99% CI. (D) Plot of apparent association rates (open circles) of Cy3-NSP1 with tethered eIF1–40S subunit complexes at the indicated NSP1…

Fig. 3.. NSP1 preferentially associated with the open head conformation of the 40S subunit. (A) Model of the human 40S subunit (gray) bound by the HCV IRES (blue) (PDB ID code 5A2Q) (38). This model of the IRES ends at the start codon (AUG, highlighted in orange), leaving the mRNA entry channel of the 40S subunit empty. Domain II of the IRES holds the head of the 40S subunit in the open conformation. (B) Schematic of the single-molecule fluorescence assay. The 40S ribosomal subunits were labeled with Cy5 dye via RACK1-ybbR. Preformed IRES–40S-Cy5 complexes were tethered to the ZMW imaging surface. At the start of data acquisition, Cy3-NSP1 (N-terminal ybbR tag) was added at 25 nM (final concentration) at 30 °C. (C) Example single-molecule fluorescence trace that depicts a tethered 40S–HCV+0 complex and subsequent association of NSP1. The 40S subunit and ybbR-NSP1 were labeled with Cy5 (red) and Cy3 (green) dyes, respectively. Loss of fluorescence signal due to dye photobleaching is indicated. Raw fluorescence intensities were corrected in this image to set baseline intensities to zero for presentation. The association time (Δt) was defined as time elapsed from the addition of Cy3-NSP1…

Fig. 4.. The mRNA within the entry channel of the 40S subunit inhibited SARS-CoV-2 NSP1 association. (A) Image of the intersubunit interface of the human 40S ribosomal subunit, with the approximate positions of mRNA (gray) and NSP1 (purple) modeled to show the predicted steric clash when segments of RNA longer than 6 nt are downstream of the start codon (blue). Models were aligned using ChimeraX (mmaker command) and PDB ID codes 6ZLW (19) and 6YAL (76). (B) Plot of the fraction of the indicated mRNA–40S subunit complexes bound at least once by SARS-CoV-2 NSP1 for ≥∼5 s. Error bars represent 99% CI. (C) Plot of the cumulative probability of observed Cy3-NSP1 association times with the indicated mRNA–40S indicates eIF1, eIF1A, eIF3Δj, eIF5, and TC(GMPPNP) were included at all stages of the experiment. Lines represent fits to single- or double-exponential functions. See SI Appendix, Table S4 for samples sizes and the parameters for relevant fits. (D) Plot of the reciprocal apparent association rates (kobs) (light blue) of SARS-CoV-2 NSP1 binding to the indicated mRNA–40S subunit complexes, derived from fits of the data to double-exponential functions, with the fast association rate reported…

Fig. 5.. NSP1 inefficiently associated with 80S ribosomes assembled on the CrPV IRES. (A) Model of the human 80S ribosome (PDB ID code 4UG0). The 40S and 60S subunits are in gray and pink, respectively. The ybbR tag was fused to either the N terminus of uS19 (green, 40S) or the C terminus of uL18 (red, 60S). Based on available structural models, the ybbR tags were predicted to be within FRET distance in translation-competent 80S ribosomes. (B) Example fluorescent trace and calculated FRET efficiency plot. The 40S-ybbR-Cy3 and 60S-ybbR-Cy5 subunits were incubated with the CrPV IRES to assemble 80S ribosomes on the biotinylated RNA. Following tethering of the complex, molecules were imaged at equilibrium using TIRFM. Molecules were expected to begin in a Cy3 (green, FRET donor) to Cy5 (red, acceptor) FRET state, followed by photobleaching of both dyes. The region of the trace that corresponds to FRET is highlighted by the gray box. (C) Plot of the distribution of observed FRET efficiencies for the intersubunit FRET signal on 80S ribosomes. Frequencies of observed FRET efficiencies were binned into 35 bins (open circles) across the indicated range. The line represents a fit to a…

Fig. 6.. NSP1 remained bound to 40S subunits upon association with model mRNAs. (A) Plot of the fraction of stable (≥5 s) 40S subunit binding events that coassociated with NSP1, as defined in B. Error bars represent 99% CI. (B) Example single-molecule fluorescence trace that depicts association of NSP1–40S subunit complexes with a tethered HCV+0 IRES molecule. The 40S subunit and NSP1 were labeled with Cy3 (green) and Cy5.5 (magenta) dyes, respectively. Raw fluorescence intensities were corrected in this image to set baseline intensities to zero for presentation. The initial NSP1–40S subunit association time (Δt1) was defined as the time elapsed from the addition of the complex until the burst of Cy3 and Cy5.5 fluorescence, which signified association of the NSP1-40S subunit complex with the tethered IRES. In experiments that lacked NSP1, Δt1 was defined using the first burst of Cy3 signal alone. The 40S subunit lifetime (τ1) was defined as the duration of the Cy3 fluorescence signal. The initial NSP1 lifetime (NSP1 τ1) was defined as the duration of the Cy5.5 signal that coappeared with the Cy3 signal. For NSP1 reassociation analyses, we focused on ZMWs where a single 40S subunit…

Fig. 7.. Proposed model. In the absence of eIF3j, NSP1 preferentially associates with the open conformation of the 40S subunit to block full accommodation of the mRNA in the entry channel, which inhibits translation initiation. How incomplete mRNA accommodation impacts mRNA recruitment, scanning, start codon selection, 60S subunit recruitment, and the transition to elongation remain open questions. Whether and how NSP1–40S subunit complexes lead to mRNA degradation is also unknown.