Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1-40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.
Highlights
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic
Using single-molecule fluorescence assays to monitor nonstructural protein 1 (NSP1)–40S subunit binding in real time, we determine that eukaryotic translation initiation factors allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of messenger RNAs (mRNAs)
We first recapitulated and quantified the extent of translation inhibition achieved by NSP1 from SARS-CoV-2
Summary
NSP1 Inhibited Translation of Host and SARS-CoV-2 Model mRNAs. We first recapitulated and quantified the extent of translation inhibition achieved by NSP1 from SARS-CoV-2. Translation of all four model viral mRNAs was reduced significantly by ∼50% upon addition of 400 nM NSP1 relative to reactions that lacked NSP1 (P ≤ 0.0008, unpaired t test) (Fig. 1C) This level of inhibition was similar to the. Translation of the 5′ UTR−3′ UTR(S) model viral mRNA was modestly increased (∼36%) relative to the host and other viral reporters in our extract-based assays (P ≤ 0.0006, one-way ANOVA) (Fig. 1C). This may suggest that enhanced translational activity of viral RNAs relative to host mRNAs could play a role in infection and evasion of NSP1 action. As predicted for a simple bimolecular interaction, NSP1 association times (Δt, the time elapsed from its addition until appearance of Cy3 signal) decreased with increasing concentration
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