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1.
Cell Rep ; 39(11): 110954, 2022 Jun 14.
Article in English | MEDLINE | ID: covidwho-1866958

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response.


Subject(s)
COVID-19 , Viral Nonstructural Proteins , Cell Line , Humans , RNA Stability , SARS-CoV-2 , Viral Nonstructural Proteins/metabolism
2.
Nat Commun ; 13(1): 2237, 2022 04 25.
Article in English | MEDLINE | ID: covidwho-1805612

ABSTRACT

The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , GATA6 Transcription Factor/genetics , Humans , Peptidyl-Dipeptidase A/metabolism , Proviruses/genetics , SARS-CoV-2/genetics
3.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-313594

ABSTRACT

At least six small alternate-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the ICTV. We recommend calling the 39 codon Spike-overlapping ORF ORF2b;the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b;the 33 codon ORF3d isoform ORF3d-2;and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity due to prior usage of alternative names.

4.
Mol Cell ; 81(11): 2261-2265, 2021 06 03.
Article in English | MEDLINE | ID: covidwho-1246090

ABSTRACT

COVID-19 altered our lives and pushed scientific research to operate at breakneck speed, leading to significant breakthroughs in record time. We asked experts in the field about the challenges they faced in transitioning, rapidly but safely, to working on the virus while navigating the shutdown. Their voices converge on the importance of teamwork, forging new collaborations, and working toward a shared goal.


Subject(s)
Biomedical Research , COVID-19/epidemiology , COVID-19/prevention & control , Pandemics , Quarantine , SARS-CoV-2 , Humans , Poetry as Topic
5.
Nature ; 594(7862): 240-245, 2021 06.
Article in English | MEDLINE | ID: covidwho-1225510

ABSTRACT

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-191. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response2,3. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression4-7, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Host-Pathogen Interactions , Protein Biosynthesis , SARS-CoV-2/pathogenicity , 5' Untranslated Regions/genetics , COVID-19/genetics , COVID-19/immunology , Cell Line , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate/genetics , Protein Biosynthesis/genetics , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/metabolism , Ribosomes/metabolism , Viral Nonstructural Proteins/metabolism
6.
Virology ; 558: 145-151, 2021 06.
Article in English | MEDLINE | ID: covidwho-1152685

ABSTRACT

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the International Committee on Taxonomy of Viruses. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity caused by prior usage of alternative names.


Subject(s)
Open Reading Frames , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus , Terminology as Topic , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/classification , Spike Glycoprotein, Coronavirus/genetics
7.
Nature ; 589(7840): 125-130, 2021 01.
Article in English | MEDLINE | ID: covidwho-752477

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic1. To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques2-4, we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.


Subject(s)
Gene Expression Profiling , Genome, Viral/genetics , Open Reading Frames/genetics , Protein Biosynthesis , SARS-CoV-2/genetics , Viral Proteins/biosynthesis , Viral Proteins/genetics , Animals , Cell Line , Humans , Molecular Sequence Annotation , Peptides/genetics , Peptides/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Ribosomes/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Viral Proteins/metabolism
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