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Mol Cell ; 80(6): 1055-1066.e6, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-1009762


The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.

COVID-19/metabolism , Protein Biosynthesis , RNA, Messenger/metabolism , RNA, Viral/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Viral Nonstructural Proteins/metabolism , Animals , COVID-19/genetics , COVID-19/pathology , Chlorocebus aethiops , Cryoelectron Microscopy , Humans , RNA, Messenger/genetics , RNA, Viral/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , Ribosome Subunits, Small, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/ultrastructure , Ribosome Subunits, Small, Eukaryotic/virology , SARS-CoV-2/genetics , SARS-CoV-2/ultrastructure , Vero Cells , Viral Nonstructural Proteins/genetics
J Gen Virol ; 102(1)2021 01.
Article in English | MEDLINE | ID: covidwho-910383


The emerging pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused social and economic disruption worldwide, infecting over 9.0 million people and killing over 469 000 by 24 June 2020. Unfortunately, no vaccine or antiviral drug that completely eliminates the transmissible disease coronavirus disease 2019 (COVID-19) has been developed to date. Given that coronavirus nonstructural protein 1 (nsp1) is a good target for attenuated vaccines, it is of great significance to explore the detailed characteristics of SARS-CoV-2 nsp1. Here, we first confirmed that SARS-CoV-2 nsp1 had a conserved function similar to that of SARS-CoV nsp1 in inhibiting host-protein synthesis and showed greater inhibition efficiency, as revealed by ribopuromycylation and Renilla luciferase (Rluc) reporter assays. Specifically, bioinformatics and biochemical experiments showed that by interacting with 40S ribosomal subunit, the lysine located at amino acid 164 (K164) was the key residue that enabled SARS-CoV-2 nsp1 to suppress host gene expression. Furthermore, as an inhibitor of host-protein expression, SARS-CoV-2 nsp1 contributed to cell-cycle arrest in G0/G1 phase, which might provide a favourable environment for virus production. Taken together, this research uncovered the detailed mechanism by which SARS-CoV-2 nsp1 K164 inhibited host gene expression, laying the foundation for the development of attenuated vaccines based on nsp1 modification.

Host-Pathogen Interactions/genetics , Lysine/genetics , Ribosomal Proteins/genetics , Ribosome Subunits, Small, Eukaryotic/genetics , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics , Amino Acid Sequence , Amino Acid Substitution , Computational Biology/methods , G1 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Lysine/metabolism , Mutation , Ribosomal Proteins/antagonists & inhibitors , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/virology , SARS Virus/genetics , SARS Virus/metabolism , SARS-CoV-2/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Viral Nonstructural Proteins/metabolism
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