Universal features of Nsp1-mediated translational shutdown by coronaviruses (preprint)

Nonstructural protein 1 (Nsp1) produced by coronaviruses shuts down host protein synthesis in infected cells. The C-terminal domain of SARS-CoV-2 Nsp1 was shown to bind to the small ribosomal subunit to inhibit translation, but it is not clear whether this mechanism is broadly used by coronaviruses, whether the N-terminal domain of Nsp1 binds the ribosome, or how Nsp1 specifically permits translation of viral mRNAs. Here, we investigated Nsp1 from three representative Betacoronaviruses - SARS-CoV-2, MERS-CoV, and Bat-Hp-CoV - using structural, biophysical, and biochemical assays. We revealed a conserved mechanism of host translational shutdown across the three coronaviruses. We further demonstrated that the N-terminal domain of Bat-Hp-CoV Nsp1 binds to the decoding center of the 40S subunit, where it would prevent mRNA and eIF1A binding. Structure-based biochemical experiments identified a conserved role of these inhibitory interactions in all three coronaviruses and showed that the same regions of Nsp1 are responsible for the preferential translation of viral mRNAs. Our results provide a mechanistic framework to understand how Betacoronaviruses overcome translational inhibition to produce viral proteins.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome (preprint)

Programmed ribosomal frameshifting is the key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream viral proteins. Here we present the cryo-EM structure of the mammalian ribosome in the process of translating viral RNA paused in a conformation primed for frameshifting. We observe that the viral RNA adopts a pseudoknot structure lodged at the mRNA entry channel of the ribosome to generate tension in the mRNA that leads to frameshifting. The nascent viral polyprotein that is being synthesized by the ribosome paused at the frameshifting site forms distinct interactions with the ribosomal polypeptide exit tunnel. We use biochemical experiments to validate our structural observations and to reveal mechanistic and regulatory features that influence the frameshifting efficiency. Finally, a compound previously shown to reduce frameshifting is able to inhibit SARS-CoV-2 replication in infected cells, establishing coronavirus frameshifting as target for antiviral intervention.

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation (preprint)

The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.Competing Interest StatementThe authors have declared no competing interest.View Full Text