ARS-CoV-2 is the causing agent of the COVID-19 pandemic and belongs to the genus of beta-coronaviruses, with enveloped, positive sense and single-stranded genomic RNA 1. On entering host cells, the viral genomic RNA is translated by the cellular protein synthesis machinery to produce a set of non-structural proteins (NSPs) 2. NSPs render the cellular conditions favorable for viral infection and viral mRNA synthesis 3. Coronaviruses have evolved specialized mechanisms to hijack the host gene expression machinery and employ cellular resources to regulate viral protein production. Such mechanisms are common for many viruses and include inhibition of host protein synthesis and endonucleolytic cleavage of host messenger RNAs (mRNAs) 4,5. In cells infected with the closely related SARS-CoV, one of the most enigmatic viral proteins is the host shutoff factor, Nsp1. Nsp1 is encoded by the gene closest to the 5′ end of the viral genome and is among the first proteins to be expressed after cell entry and infection to repress multiple steps of host protein expression 6-9. Initial structural characterization of the isolated SARS-CoV Nsp1 protein revealed the structure of its N-terminal domain, whereas its C-terminal region was flexibly disordered 10. Furthermore, it was shown that SARS-CoV Nsp1 suppresses host innate immune functions, mainly by targeting type I interferon expression and antiviral signaling pathways 11. Taken together, Nsp1 serves as a potential virulence factor for coronaviruses and represents an attractive target for live attenuated vaccine development 12,13. To provide molecular insights into the mechanism of SARS-CoV-2 Nsp1-mediated translation inhibition, we solved the structures of ribosomal complexes isolated from HEK293 lysates supplemented with recombinant purified Nsp1 as well as of an in vitro reconstituted 40S-Nsp1 complex using cryo-EM. We complement our findings by reporting in vitro and in vivo translation inhibition in the presence of Nsp1 that is relieved after mutating key interacting residues. Furthermore, we show that the translation output of reporters containing full-length viral 5′ untranslated regions (UTRs) is significantly enhanced, which could explain how Nsp1 inhibits global translation while still translating sufficient amounts of viral mRNAs. Results C-terminal domain of SARS-CoV-2 Nsp1 binds to the mRNA entry channel. To elucidate the mechanism of how Nsp1 inhibits translation, we aimed to identify the structures of potential ribosomal complexes as binding targets (Fig. 1). Previously, it has been suggested that Nsp1 mainly targets the ribosome at the translation initiation step 9. We thus treated lysed HEK293E cells with bacterially expressed and purified Nsp1 and loaded the cleared lysate on a sucrose gradient. Fractions containing ribosomal particles were then analyzed for the presence of Nsp1. Interestingly, Nsp1 not only co-migrated with 40S particles, but also with 80S ribosomal complexes (Fig. 1c), suggesting that it interacts with a range of different ribosomal states. W...

AbstractThe 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.

Programmed ribosomal frameshifting is a key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal mRNA channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

Final maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA Using cryo-electron microscopy (cryo-EM), we have determined the structure of a yeast cytoplasmic pre-40S particle in complex with Enp1, Ltv1, Rio2, Tsr1, and Pno1 assembly factors poised to initiate final maturation. The structure reveals that the pre-rRNA adopts a highly distorted conformation of its 3' major and 3' minor domains stabilized by the binding of the assembly factors. This observation is consistent with a mechanism that involves concerted release of the assembly factors orchestrated by the folding of the rRNA in the head of the pre-40S subunit during the final stages of maturation. Our results provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle toward the mature form capable of engaging in translation.

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.