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Science ; 372(6548): 1306-1313, 2021 06 18.
Article in English | MEDLINE | ID: covidwho-1228853


Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows 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 messenger RNA (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.

Frameshifting, Ribosomal , RNA, Viral/genetics , Ribosomes/ultrastructure , SARS-CoV-2/genetics , Viral Proteins/biosynthesis , Animals , Antiviral Agents/pharmacology , Codon, Terminator , Coronavirus RNA-Dependent RNA Polymerase/biosynthesis , Coronavirus RNA-Dependent RNA Polymerase/chemistry , Coronavirus RNA-Dependent RNA Polymerase/genetics , Cryoelectron Microscopy , Fluoroquinolones/pharmacology , Frameshifting, Ribosomal/drug effects , Genome, Viral , Humans , Image Processing, Computer-Assisted , Models, Molecular , Nucleic Acid Conformation , Open Reading Frames , Protein Folding , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Viral Proteins/chemistry , Viral Proteins/genetics , Virus Replication/drug effects
Nat Struct Mol Biol ; 27(11): 1094, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-882915


An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nat Struct Mol Biol ; 27(10): 959-966, 2020 10.
Article in English | MEDLINE | ID: covidwho-752470


The SARS-CoV-2 non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, suppresses host innate immune functions. By combining cryo-electron microscopy and biochemistry, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes, including the 43S pre-initiation complex and the non-translating 80S ribosome. The protein inserts its C-terminal domain into the mRNA channel, where it interferes with mRNA binding. We observe translation inhibition in the presence of Nsp1 in an in vitro translation system and in 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 global inhibition of translation by Nsp1 with efficient translation of the viral mRNA to allow expression of viral genes.

Betacoronavirus/chemistry , Betacoronavirus/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , 5' Untranslated Regions , Betacoronavirus/genetics , Cryoelectron Microscopy , HEK293 Cells , HeLa Cells , Host-Pathogen Interactions/physiology , Humans , Models, Molecular , Mutation , Protein Conformation , Protein Domains , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/genetics