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2.
Cell Host Microbe ; 30(3): 373-387.e7, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1767977

ABSTRACT

SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
4.
Cell Rep ; 38(10): 110434, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1729611

ABSTRACT

Type I interferons (IFN-I) are essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses. However, few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue. We identified the RNA helicase DHX16 as a potential pattern recognition receptor (PRR). Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. These effects extended to members of other virus families, including Zika and SARS-CoV-2. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3-Ub ligase TRIM6. DHX16 recognizes a signal in influenza RNA segments that undergo splicing and requires its RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a PRR that partners with RIG-I for optimal activation of antiviral immunity requiring unanchored poly-Ub.


Subject(s)
DEAD Box Protein 58 , Interferon Type I , RNA Helicases , RNA, Viral , Receptors, Immunologic , Zika Virus Infection , Zika Virus , COVID-19 , DEAD Box Protein 58/immunology , Humans , Immunity, Innate , Interferon Type I/immunology , RNA Helicases/immunology , Receptors, Immunologic/immunology , SARS-CoV-2 , Tripartite Motif Proteins , Zika Virus/genetics , Zika Virus Infection/immunology
5.
Nature ; 602(7897): 487-495, 2022 02.
Article in English | MEDLINE | ID: covidwho-1585830

ABSTRACT

The emergence of SARS-CoV-2 variants of concern suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on the characterization of changes in the spike protein in variants of concern, mutations outside of spike are likely to contribute to adaptation. Here, using unbiased abundance proteomics, phosphoproteomics, RNA sequencing and viral replication assays, we show that isolates of the Alpha (B.1.1.7) variant3 suppress innate immune responses in airway epithelial cells more effectively than first-wave isolates. We found that the Alpha variant has markedly increased subgenomic RNA and protein levels of the nucleocapsid protein (N), Orf9b and Orf6-all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein that is required for activation of the RNA-sensing adaptor MAVS. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful transmission of the Alpha variant, and may increase in vivo replication and duration of infection4. The importance of mutations outside the spike coding region in the adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the N and Orf9b regulatory regions of the Delta and Omicron variants.


Subject(s)
COVID-19/immunology , COVID-19/virology , Evolution, Molecular , Immune Evasion , Immunity, Innate/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , COVID-19/transmission , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Immunity, Innate/genetics , Interferons/immunology , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Proteomics , RNA, Viral/genetics , RNA-Seq , SARS-CoV-2/classification , SARS-CoV-2/growth & development
7.
mSystems ; 6(5): e0038821, 2021 Oct 26.
Article in English | MEDLINE | ID: covidwho-1406606

ABSTRACT

Current epidemics, such as AIDS or flu, and the emergence of new threatening pathogens, such as the one causing the current coronavirus disease 2019 (COVID-19) pandemic, represent major global health challenges. While vaccination is an important part of the arsenal to counter the spread of viral diseases, it presents limitations and needs to be complemented by efficient therapeutic solutions. Intricate knowledge of host-pathogen interactions is a powerful tool to identify host-dependent vulnerabilities that can be exploited to dampen viral replication. Such host-directed antiviral therapies are promising and are less prone to the development of drug-resistant viral strains. Here, we first describe proteomics-based strategies that allow the rapid characterization of host-pathogen interactions. We then discuss how such data can be exploited to help prioritize compounds with potential host-directed antiviral activity that can be tested in preclinical models.

8.
Science ; 373(6554): 541-547, 2021 07 30.
Article in English | MEDLINE | ID: covidwho-1334531

ABSTRACT

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning , Lipidoses/chemically induced , Phospholipids/metabolism , SARS-CoV-2/drug effects , A549 Cells , Animals , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Antiviral Agents/toxicity , COVID-19/virology , Cations , Chlorocebus aethiops , Dose-Response Relationship, Drug , Female , Humans , Mice , Microbial Sensitivity Tests , SARS-CoV-2/physiology , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacology , Surface-Active Agents/toxicity , Vero Cells , Virus Replication/drug effects
9.
Curr Biol ; 31(16): 3504-3514.e9, 2021 08 23.
Article in English | MEDLINE | ID: covidwho-1281407

ABSTRACT

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has emphasized the vulnerability of human populations to novel viral pressures, despite the vast array of epidemiological and biomedical tools now available. Notably, modern human genomes contain evolutionary information tracing back tens of thousands of years, which may help identify the viruses that have impacted our ancestors-pointing to which viruses have future pandemic potential. Here, we apply evolutionary analyses to human genomic datasets to recover selection events involving tens of human genes that interact with coronaviruses, including SARS-CoV-2, that likely started more than 20,000 years ago. These adaptive events were limited to the population ancestral to East Asian populations. Multiple lines of functional evidence support an ancient viral selective pressure, and East Asia is the geographical origin of several modern coronavirus epidemics. An arms race with an ancient coronavirus, or with a different virus that happened to use similar interactions as coronaviruses with human hosts, may thus have taken place in ancestral East Asian populations. By learning more about our ancient viral foes, our study highlights the promise of evolutionary information to better predict the pandemics of the future. Importantly, adaptation to ancient viral epidemics in specific human populations does not necessarily imply any difference in genetic susceptibility between different human populations, and the current evidence points toward an overwhelming impact of socioeconomic factors in the case of coronavirus disease 2019 (COVID-19).


Subject(s)
Coronavirus Infections/history , Coronavirus/genetics , Genome, Human/genetics , Host Microbial Interactions/genetics , Pandemics/history , Coronavirus Infections/virology , Datasets as Topic , Evolution, Molecular , Far East/epidemiology , Gene Frequency , Genetic Predisposition to Disease , Genome, Viral/genetics , Genome-Wide Association Study , History, Ancient , Human Genome Project , Humans , Mutation , Phylogeny , Selection, Genetic
10.
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
11.
Cell ; 184(9): 2271-2275, 2021 04 29.
Article in English | MEDLINE | ID: covidwho-1149106

ABSTRACT

The scientific world rewards the individual while often discouraging collaboration. However, times of crisis show us how much more we can accomplish when we work together. Here, we describe our approach to breaking down silos and fostering global collaborations and share the lessons we have learned, especially pertaining to research on SARS-CoV-2.


Subject(s)
Cooperative Behavior , Science , COVID-19/virology , Communication , Humans , Internationality , Research Support as Topic , SARS-CoV-2/physiology
12.
Cell ; 184(1): 106-119.e14, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1064913

ABSTRACT

The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.


Subject(s)
COVID-19/genetics , Coronavirus Infections/genetics , Coronavirus/physiology , Genome-Wide Association Study , Host-Pathogen Interactions , SARS-CoV-2/physiology , A549 Cells , Animals , Biosynthetic Pathways/drug effects , COVID-19/virology , Cell Line , Chlorocebus aethiops , Cholesterol/biosynthesis , Cholesterol/metabolism , Cluster Analysis , Clustered Regularly Interspaced Short Palindromic Repeats , Common Cold/genetics , Common Cold/virology , Coronavirus/classification , Coronavirus Infections/virology , Gene Knockout Techniques , Host-Pathogen Interactions/drug effects , Humans , Mice , Phosphatidylinositols/biosynthesis , Vero Cells , Virus Internalization/drug effects , Virus Replication
13.
Science ; 371(6532): 926-931, 2021 02 26.
Article in English | MEDLINE | ID: covidwho-1048642

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Depsipeptides/pharmacology , Peptide Elongation Factor 1/antagonists & inhibitors , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adenosine Monophosphate/therapeutic use , Alanine/analogs & derivatives , Alanine/pharmacology , Alanine/therapeutic use , Animals , Antiviral Agents/therapeutic use , COVID-19/prevention & control , COVID-19/virology , Coronavirus Nucleocapsid Proteins/biosynthesis , Coronavirus Nucleocapsid Proteins/genetics , Depsipeptides/administration & dosage , Depsipeptides/therapeutic use , Drug Evaluation, Preclinical , Female , HEK293 Cells , Humans , Lung/virology , Mice, Inbred C57BL , Mutation , Peptides, Cyclic , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Virus Replication/drug effects
14.
J Proteome Res ; 20(2): 1133-1152, 2021 02 05.
Article in English | MEDLINE | ID: covidwho-1036024

ABSTRACT

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), was declared a pandemic infection in March 2020. As of December 2020, two COVID-19 vaccines have been authorized for emergency use by the U.S. Food and Drug Administration, but there are no effective drugs to treat COVID-19, and pandemic mitigation efforts like physical distancing have had acute social and economic consequences. In this perspective, we discuss how the proteomic research community can leverage technologies and expertise to address the pandemic by investigating four key areas of study in SARS-CoV-2 biology. Specifically, we discuss how (1) mass spectrometry-based structural techniques can overcome limitations and complement traditional structural approaches to inform the dynamic structure of SARS-CoV-2 proteins, complexes, and virions; (2) virus-host protein-protein interaction mapping can identify the cellular machinery required for SARS-CoV-2 replication; (3) global protein abundance and post-translational modification profiling can characterize signaling pathways that are rewired during infection; and (4) proteomic technologies can aid in biomarker identification, diagnostics, and drug development in order to monitor COVID-19 pathology and investigate treatment strategies. Systems-level high-throughput capabilities of proteomic technologies can yield important insights into SARS-CoV-2 biology that are urgently needed during the pandemic, and more broadly, can inform coronavirus virology and host biology.


Subject(s)
COVID-19/prevention & control , Proteome/metabolism , Proteomics/methods , SARS-CoV-2/metabolism , COVID-19/epidemiology , COVID-19/virology , Host-Pathogen Interactions , Humans , Mass Spectrometry/methods , Pandemics , Protein Interaction Maps , Protein Processing, Post-Translational , SARS-CoV-2/physiology , Viral Proteins/metabolism
15.
Science ; 370(6523): 1473-1479, 2020 12 18.
Article in English | MEDLINE | ID: covidwho-913670

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , Antibody Affinity , Chlorocebus aethiops , Cryoelectron Microscopy , Humans , Neutralization Tests , Protein Binding , Protein Stability , Single-Domain Antibodies/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
16.
Proc Natl Acad Sci U S A ; 117(45): 28344-28354, 2020 11 10.
Article in English | MEDLINE | ID: covidwho-887237

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.


Subject(s)
COVID-19/metabolism , Interferons/metabolism , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Viral Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Binding Sites , Chlorocebus aethiops , HEK293 Cells , Humans , Nuclear Matrix-Associated Proteins/chemistry , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/metabolism , Protein Binding , Signal Transduction , Vero Cells
17.
Cancer Discov ; 10(7): 916-921, 2020 07.
Article in English | MEDLINE | ID: covidwho-342772

ABSTRACT

The mapping of SARS-CoV-2 human protein-protein interactions by Gordon and colleagues revealed druggable targets that are hijacked by the virus. Here, we highlight several oncogenic pathways identified at the host-virus interface of SARS-CoV-2 to enable cancer biologists to apply their knowledge for rapid drug repurposing to treat COVID-19, and help inform the response to potential long-term complications of the disease.


Subject(s)
Betacoronavirus , Coronavirus Infections/drug therapy , Drug Repositioning , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Pneumonia, Viral/drug therapy , COVID-19 , Cell Cycle , Coronavirus Infections/genetics , Coronavirus Infections/metabolism , Coronavirus Infections/physiopathology , DNA Damage , Epigenomics , Humans , Neoplasm Proteins/drug effects , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/physiopathology , Pandemics , Pneumonia, Viral/genetics , Pneumonia, Viral/metabolism , Pneumonia, Viral/physiopathology , Protein Biosynthesis , SARS-CoV-2
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