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

Abstract

SARS-CoV-2 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. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also 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 demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we 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 associates 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.

SARS-CoV-2 utilizes a multipronged strategy to suppress host protein synthesis

“…Still, how the virus can overcome the NSP1-mediated block of translation and how NSP1 effects come into play during infection remained unclear. We reveal here that in resemblance to what had been described for SARS-CoV NSP1, SARS-CoV-2 NSP1 induces shutdown of host protein translation by two mechanisms: first, it stalls canonical mRNA translation as was reported by others [24][25][26][27][28]41 leading to general reduction in the translation capacity of infected cells. Second, NSP1 leads to accelerated cellular mRNA degradation.…”

“…By their nature, deep sequencing measurements provide relative values but not absolute quantification of RNA and translation levels. Since SARS-CoV-2 encoded protein, NSP1, was recently shown to interfere with translation by blocking the mRNA entry channel of ribosomes [25][26][27][28] , and since the extent to which SARS-CoV-2 interferes with the overall levels of cellular mRNA was not assessed, we next examined if SARS-CoV-2 infection affects global translation and RNA levels. To quantify absolute translation levels, we measured nascent protein synthesis levels using an analogue of puromycin, O-Propargyl Puromycin (OPP), which incorporates into elongating polypeptide chains 34 .…”

“…In CoVs, the most prominent and well characterized cellular shutoff protein is NSP1 23 . So far, studies on SARS-CoV-2 NSP1 demonstrated it restricts translation by directly binding to the ribosome 40S subunit [25][26][27][28] , thereby globally inhibiting translation initiation. For SARS-CoV, on top of this translation effect, NSP1 interactions with the 40S was also shown to induce cleavage of translated cellular mRNAs, thereby accelerating their turnover their translation we compiled a list of 14 long non-coding RNAs (lncRNAs) that localize to the cytoplasm and are well expressed in our data but, as expected, poorly translated ( Figure S4B).…”

“…Indeed, several studies focused on SARS-CoV-2 encoded NSP1, showing it binds to the 40S ribosomal subunit, leading to reduction in mRNA translation both in vitro and in cells [24][25][26][27][28] . Still, how the virus can overcome the NSP1-mediated block of translation and how NSP1 effects come into play during infection remained unclear.…”

“…NSP1 is the best characterized and most prominent coronavirus host shutoff factor 23 . Several recent studies showed SARS-CoV-2 NSP1 binds the 40s ribosome and inhibits translation [24][25][26][27][28] .…”