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1.
Nucleic Acids Res ; 50(5): 2509-2521, 2022 03 21.
Article in English | MEDLINE | ID: covidwho-1722548

ABSTRACT

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.


Subject(s)
COVID-19/immunology , Immunity, Innate/immunology , RNA Editing/immunology , SARS-CoV-2/immunology , Adenosine Deaminase/genetics , Adenosine Deaminase/immunology , Adenosine Deaminase/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Base Sequence , Binding Sites/genetics , COVID-19/genetics , COVID-19/virology , Evolution, Molecular , Gene Expression/immunology , Humans , Immunity, Innate/genetics , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-Induced Helicase, IFIH1/metabolism , Mutation , Protein Binding , RNA Editing/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
2.
Elife ; 112022 01 21.
Article in English | MEDLINE | ID: covidwho-1643864

ABSTRACT

Background: Variants in IFIH1, a gene coding the cytoplasmatic RNA sensor MDA5, regulate the response to viral infections. We hypothesized that IFIH1 rs199076 variants would modulate host response and outcome after severe COVID-19. Methods: Patients admitted to an intensive care unit (ICU) with confirmed COVID-19 were prospectively studied and rs1990760 variants determined. Peripheral blood gene expression, cell populations, and immune mediators were measured. Peripheral blood mononuclear cells from healthy volunteers were exposed to an MDA5 agonist and dexamethasone ex-vivo, and changes in gene expression assessed. ICU discharge and hospital death were modeled using rs1990760 variants and dexamethasone as factors in this cohort and in-silico clinical trials. Results: About 227 patients were studied. Patients with the IFIH1 rs1990760 TT variant showed a lower expression of inflammation-related pathways, an anti-inflammatory cell profile, and lower concentrations of pro-inflammatory mediators. Cells with TT variant exposed to an MDA5 agonist showed an increase in IL6 expression after dexamethasone treatment. All patients with the TT variant not treated with steroids survived their ICU stay (hazard ratio [HR]: 2.49, 95% confidence interval [CI]: 1.29-4.79). Patients with a TT variant treated with dexamethasone showed an increased hospital mortality (HR: 2.19, 95% CI: 1.01-4.87) and serum IL-6. In-silico clinical trials supported these findings. Conclusions: COVID-19 patients with the IFIH1 rs1990760 TT variant show an attenuated inflammatory response and better outcomes. Dexamethasone may reverse this anti-inflammatory phenotype. Funding: Centro de Investigación Biomédica en Red (CB17/06/00021), Instituto de Salud Carlos III (PI19/00184 and PI20/01360), and Fundació La Marató de TV3 (413/C/2021).


Patients with severe COVID-19 often need mechanical ventilation to help them breathe and other types of intensive care. The outcome for many of these patients depends on how their immune system reacts to the infection. If the inflammatory response triggered by the immune system is too strong, this can cause further harm to the patient. One gene that plays an important role in inflammation is IFIH1 which encodes a protein that helps the body to recognize viruses. There are multiple versions of this gene which each produce a slightly different protein. It is possible that this variation impacts how the immune system responds to the virus that causes COVID-19. To investigate, Amado-Rodríguez, Salgado del Riego et al. analyzed the IFIH1 gene in 227 patients admitted to an intensive care unit in Spain for severe COVID-19 between March and December 2020. They found that patients with a specific version of the gene called TT experienced less inflammation and were more likely to survive the infection. Physicians typically treat patients with moderate to severe COVID-19 with corticosteroid drugs that reduce the inflammatory response. However, Amado-Rodríguez, Salgado del Riego et al. found that patients with the TT version of the IFIH1 gene were at greater risk of dying if they received corticosteroids. The team then applied the distribution of IFIH1 variants among different ethnic ancestries to data from a previous clinical trial, and simulated the effects of corticosteroid treatment. This 'mock' clinical trial supported their findings from the patient-derived data, which were also validated by laboratory experiments on immune cells from individuals with the TT gene. The work by Amado-Rodríguez, Salgado del Riego et al. suggests that while corticosteroids benefit some patients, they may cause harm to others. However, a real-world clinical trial is needed to determine whether patients with the TT version of the IFIH1 gene would do better without steroids.


Subject(s)
COVID-19/genetics , Inflammation/genetics , Interferon-Induced Helicase, IFIH1/genetics , SARS-CoV-2/pathogenicity , Aged , COVID-19/complications , Critical Illness , DEAD-box RNA Helicases/metabolism , Female , Humans , Inflammation/metabolism , Male , Middle Aged
3.
Genes (Basel) ; 13(1)2021 12 23.
Article in English | MEDLINE | ID: covidwho-1580896

ABSTRACT

ADAR1-mediated deamination of adenosines in long double-stranded RNAs plays an important role in modulating the innate immune response. However, recent investigations based on metatranscriptomic samples of COVID-19 patients and SARS-COV-2-infected Vero cells have recovered contrasting findings. Using RNAseq data from time course experiments of infected human cell lines and transcriptome data from Vero cells and clinical samples, we prove that A-to-G changes observed in SARS-COV-2 genomes represent genuine RNA editing events, likely mediated by ADAR1. While the A-to-I editing rate is generally low, changes are distributed along the entire viral genome, are overrepresented in exonic regions, and are (in the majority of cases) nonsynonymous. The impact of RNA editing on virus-host interactions could be relevant to identify potential targets for therapeutic interventions.


Subject(s)
Adenosine Deaminase/genetics , COVID-19/genetics , Genome, Viral , Host-Pathogen Interactions/genetics , RNA Editing , RNA, Viral/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Adenosine/metabolism , Adenosine Deaminase/immunology , Animals , COVID-19/metabolism , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , DEAD Box Protein 58/genetics , DEAD Box Protein 58/immunology , Deamination , Epithelial Cells/immunology , Epithelial Cells/virology , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Inosine/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-beta/genetics , Interferon-beta/immunology , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA, Viral/immunology , RNA-Binding Proteins/immunology , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Transcriptome , Vero Cells
4.
Nat Commun ; 12(1): 6668, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1526076

ABSTRACT

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.


Subject(s)
Adenosine Triphosphate/metabolism , Inflammation/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Mutation, Missense , RNA, Double-Stranded/metabolism , RNA, Viral/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/ultrastructure , Cryoelectron Microscopy , HEK293 Cells , Humans , Immunity, Innate/genetics , Inflammation/genetics , Interferon-Induced Helicase, IFIH1/chemistry , Interferon-Induced Helicase, IFIH1/genetics , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Conformation , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/genetics , RNA, Viral/genetics
5.
Acc Chem Res ; 54(21): 4012-4023, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1483069

ABSTRACT

In vitro-transcribed RNAs are emerging as new biologics for therapeutic innovation, as exemplified by their application recently in SARS-CoV-2 vaccinations. RNAs prepared by in vitro transcription (IVT) allow transient expression of proteins of interest, conferring safety over DNA- or virus-mediated gene delivery systems. However, in vitro-transcribed RNAs should be used with caution because of their immunogenicity, which is in part triggered by double-stranded RNA (dsRNA) byproducts during IVT. Cellular innate immune response to dsRNA byproducts can lead to undesirable consequences, including suppression of protein synthesis and cell death, which in turn can detrimentally impact the efficacy of mRNA therapy. Thus, it is critical to understand the nature of IVT byproducts and the mechanisms by which they trigger innate immune responses.Our lab has been investigating the mechanisms by which the innate immune system discriminates between "self" and "nonself" RNA, with the focus on the cytoplasmic dsRNA receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated 5 (MDA5). We have biochemically and structurally characterized critical events involving RNA discrimination and signal transduction by RIG-I or MDA5. We have used in vitro-transcribed RNAs as tools to investigate RNA specificity of RIG-I and MDA5, which required optimization of the IVT reaction and purification processes to eliminate the effect of IVT byproducts. In this Account, we summarize our current understanding of RIG-I and MDA5 and IVT reactions and propose future directions for improving IVT as a method to generate both research tools and therapeutics. Other critical proteins in cellular innate immune response to dsRNAs are also discussed. We arrange the contents in the following order: (i) innate immunity sensors for nonself RNA, including the RIG-I-like receptors (RLRs) in the cytosol and the toll-like receptors (TLRs) in the endosome, as well as cytoplasmic dsRNA-responding proteins, including protein kinase R (PKR) and 2',5'-oligoadenylate synthetases (OASes), illustrating the feature of protein-RNA binding and its consequences; (ii) the immunogenicity of IVT byproducts, specifically the generation of dsRNA molecules during IVT; and (iii) methods to reduce IVT RNA immunogenicity, including optimizations of RNA polymerases, reagents, and experimental conditions during IVT and subsequent purification.


Subject(s)
RNA, Viral/immunology , SARS-CoV-2/genetics , DEAD Box Protein 58/genetics , DEAD Box Protein 58/immunology , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , RNA, Viral/genetics , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , SARS-CoV-2/immunology
6.
Indian J Med Res ; 153(5&6): 677-683, 2021 05.
Article in English | MEDLINE | ID: covidwho-1413141

ABSTRACT

Background & objectives: Upper respiratory mucosa is the entryway for SARS-CoV-2, and cells at this site form the first line of resistance against the pathogens. Innate immune response at this point is crucial for managing the replication and early stage symptoms of virus infection. This study was aimed to evaluate the expression of pattern recognition receptors and cytokines in upper airway cells of SARS-CoV-2 infected patients. Methods: Forty seven nasopharyngeal swab (NPS) specimens from 25 SARS-CoV-2 infected patients and 22 SARS-CoV-2 negative individuals were investigated for expression of toll-like receptors (TLRs), melanoma differentiation-associated protein 5 (MDA5), NOD-like receptors family pyrin domain containing 3 (NLRP3), angiotensin-converting enzyme 2 (ACE2), interleukin (IL) - 6, tumour necrosis factor alpha (TNFα) and type-1 interferons (IFNs) by real time reverse transcription polymerase chain reaction. Results: Increased expression of TLR2, MDA5 and ACE2 was detected in SARS-CoV-2 infected patients in comparison with controls. MDA5 expression was significantly higher in asymptomatic and mildly symptomatic SARS-CoV-2 positive patients than the patients with severe symptoms. The asymptomatic group showed significant induction of type 1 IFNs than the symptomatic group. Non-specific induction of TLR7 could be observed in nasopharyngeal (NP) cells irrespective of symptoms and SARS-CoV-2 positivity. Interpretation & conclusions: The findings suggest that increased MDA5 in NP cells of asymptomatic SARS-CoV-2 positive patients may subsequently induce type 1 IFNs to protect the individuals from further clinical severity of SARS-CoV-2 infection. A future prospective study in NPS of larger cohort needs to be performed to confirm our findings.


Subject(s)
Immunity, Innate , SARS-CoV-2 , COVID-19 , Cytokines/genetics , Humans , Immunity, Innate/genetics , Interferon-Induced Helicase, IFIH1/genetics
7.
Mil Med Res ; 8(1): 49, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1398883

ABSTRACT

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses. Herein we investigate their functions in human epithelial cells, the primary and initial target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A deficiency in MDA5, RIG-I or mitochondrial antiviral signaling protein (MAVS) enhanced viral replication. The expression of the type I/III interferon (IFN) during infection was impaired in MDA5-/- and MAVS-/-, but not in RIG-I-/-, when compared to wild type (WT) cells. The mRNA level of full-length angiotensin-converting enzyme 2 (ACE2), the cellular entry receptor for SARS-CoV-2, was ~ 2.5-fold higher in RIG-I-/- than WT cells. These data demonstrate MDA5 as the predominant SARS-CoV-2 sensor, IFN-independent induction of ACE2 and anti-SARS-CoV-2 role of RIG-I in epithelial cells.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , COVID-19/immunology , DEAD Box Protein 58/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Receptors, Immunologic/metabolism , SARS-CoV-2/physiology , Adaptor Proteins, Signal Transducing/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cell Line , DEAD Box Protein 58/genetics , Humans , Interferon Type I/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Interferons/metabolism , Receptors, Immunologic/genetics , Signal Transduction , Virus Replication
8.
J Med Virol ; 93(9): 5376-5389, 2021 09.
Article in English | MEDLINE | ID: covidwho-1363676

ABSTRACT

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.


Subject(s)
DEAD Box Protein 58/immunology , Immune Evasion/genetics , Interferons/immunology , Nucleotidyltransferases/immunology , Receptors, Immunologic/immunology , SARS-CoV-2/immunology , Toll-Like Receptor 3/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/immunology , Animals , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/genetics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferons/genetics , Membrane Proteins/genetics , Membrane Proteins/immunology , Nucleotidyltransferases/genetics , Phosphoproteins/genetics , Phosphoproteins/immunology , Plasmids/chemistry , Plasmids/metabolism , /immunology , Receptors, Immunologic/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 3/genetics , Transfection , Vero Cells , Virus Replication/immunology
9.
Front Immunol ; 12: 700926, 2021.
Article in English | MEDLINE | ID: covidwho-1305649

ABSTRACT

RIG-I-like receptors (RLR), RIG-I and MDA5, are cytoplasmic viral RNA sensors that recognize viral double-stranded RNAs and trigger signals to induce antiviral responses, including type I interferon production. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) caused the coronavirus disease 2019 pandemic. However, the RLR role in innate immune response to SARS-CoV-2 has not been fully elucidated. Here, we studied the roles of RLR in cytokine expression responding to SARS-CoV-2 and found that not only MDA5 but also RIG-I are involved in innate immune responses in some types of human cells. Transfection of total RNAs extracted from SARS-CoV-2-infected cells into epithelial cells induced IFN-ß, IP-10, and Ccl5 mRNA expression. The cytokine expression was reduced by knockout of either RIG-I or MDA5, suggesting that both proteins are required for appropriate innate immune response to SARS-CoV-2. Two viral genomic RNA regions strongly induced type I IFN expression, and a 200-base fragment of viral RNA preferentially induced type I IFN in a RIG-I-dependent manner. In contrast, SARS-CoV-2 infectious particles hardly induced cytokine expression, suggesting viral escape from the host response. Viral 9b protein inhibited RIG-I and MAVS interaction, and viral 7a protein destabilized the TBK1 protein, leading to attenuated IRF-3 phosphorylation required for type I IFN expression. Our data elucidated the mechanism underlying RLR-mediated response to SARS-CoV-2 infection and viral escape from the host innate immune response.


Subject(s)
COVID-19/immunology , Interferon-Induced Helicase, IFIH1/metabolism , Receptors, Retinoic Acid/metabolism , SARS-CoV-2/physiology , Severe Acute Respiratory Syndrome/immunology , Gene Knockdown Techniques , HEK293 Cells , Host-Pathogen Interactions , Humans , Immune Evasion , Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Interferon Type I/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Phosphorylation , RNA, Viral/immunology , Receptors, Retinoic Acid/genetics , Signal Transduction , Viral Matrix Proteins/metabolism
10.
Biochem J ; 478(10): 1853-1859, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1232077

ABSTRACT

The current SARS-CoV-2 pandemic has spurred new interest in interferon signaling in response to viral pathogens. Much of what we know about the signaling molecules and associated signal transduction induced during the host cellular response to viral pathogens has been gained from research conducted from the 1990's to the present day, but certain intricacies of the mechanisms involved, still remain unclear. In a recent study by Vaughn et al. the authors examine one of the main mechanisms regulating interferon induction following viral infection, the RIG-I/MAVS/IRF3 pathway, and find that similar to PKR both DICER interacting proteins, PACT and TRBP, regulate RIG-I signaling in an opposing manner. More specifically, the reported findings demonstrate, like others, that PACT stimulates RIG-I-mediated signaling in a manner independent of PACT dsRNA-binding ability or phosphorylation at sites known to be important for PACT-dependent PKR activation. In contrast, they show for the first time that TRBP inhibits RIG-I-mediated signaling. RIG-I inhibition by TRBP did not require phosphorylation of sites shown to be important for inhibiting PKR, nor did it involve PACT or PKR, but it did require the dsRNA-binding ability of TRBP. These findings open the door to a complex co-regulation of RIG-I, PKR, MDA5, miRNA processing, and interferon induction.


Subject(s)
COVID-19/immunology , Interferons/metabolism , SARS-CoV-2/immunology , Signal Transduction/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , COVID-19/virology , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , Gene Expression Regulation/immunology , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/metabolism , Interferons/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Nuclear Receptor Coactivators/genetics , Nuclear Receptor Coactivators/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism
11.
J Med Virol ; 93(9): 5376-5389, 2021 09.
Article in English | MEDLINE | ID: covidwho-1206842

ABSTRACT

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to the pathogenesis of coronavirus disease 2019 (COVID-19). The strategy used by SARS-CoV-2 to evade antiviral immunity needs further investigation. Here, we reported that SARS-CoV-2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS-CoV-2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS-CoV-2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA-sensing pathways of RIG-I/MDA5-MAVS signaling, including RIG-I, MDA-5, MAVS, TBK1, and IKKε, rather than IRF3-5D, which is the active form of IRF3. SARS-CoV-2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA-sensing pathway of TLR3-TRIF signaling and the adaptor protein of the cytosolic DNA-sensing pathway of cGAS-STING signaling, respectively. A mechanistic analysis revealed that the SARS-CoV-2 ORF9b protein interacted with RIG-I, MDA-5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS-CoV-2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS-CoV-2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS-CoV-2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID-19.


Subject(s)
DEAD Box Protein 58/immunology , Immune Evasion/genetics , Interferons/immunology , Nucleotidyltransferases/immunology , Receptors, Immunologic/immunology , SARS-CoV-2/immunology , Toll-Like Receptor 3/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/immunology , Animals , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/genetics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferons/genetics , Membrane Proteins/genetics , Membrane Proteins/immunology , Nucleotidyltransferases/genetics , Phosphoproteins/genetics , Phosphoproteins/immunology , Plasmids/chemistry , Plasmids/metabolism , /immunology , Receptors, Immunologic/genetics , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 3/genetics , Transfection , Vero Cells , Virus Replication/immunology
12.
Int Immunopharmacol ; 96: 107671, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1227634

ABSTRACT

Chlorogenic acid (CGA) is a phenolic compound that has been well studied for its antiviral, anti-inflammatory and immune stimulating properties. This research was aimed to focus on the antiviral properties of CGA on infectious bronchitis virus (IBV) in vivo and in vitro for the very first time. The outcome of in vitro experiments validated that, out of five previously reported antiviral components, CGA significantly reduced the relative mRNA expression of IBV-N in CEK cells. At high concentration (400 mg/kg), CGA supplementation reduced IBV-N mRNA expression levels and ameliorated the injury in trachea and lungs. The mRNA expression levels of IL-6, IL-1ß, IL-12, and NF-κB were considerably turned down, but IL-22 and IL-10 were enhanced in trachea. However, CGA-H treatment had considerably increased the expression levels of MDA5, MAVS, TLR7, MyD88, IRF7, IFN-ß and IFN-α both in trachea and lungs. Moreover, CGA-H notably induced the CD3+, CD3+ CD4+ and CD4+/CD8+ proliferation and significantly increased the IgA, IgG, and IgM levels in the serum. In conclusion, these results showed that at high concentration CGA is a strong anti-IBV compound that can effectively regulate the innate immunity through MDA5, TLR7 and NF-κB signaling pathways and have the potential to induce the cell mediated and humoral immune response in IBV infected chickens.


Subject(s)
Chlorogenic Acid/pharmacology , Coronavirus Infections/drug therapy , Gammacoronavirus/drug effects , Interferon-Induced Helicase, IFIH1/metabolism , NF-kappa B/metabolism , Toll-Like Receptor 7/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Antiviral Agents/pharmacology , Cells, Cultured , Chickens , Coronavirus Infections/immunology , Coronavirus Infections/virology , Disease Models, Animal , Gammacoronavirus/immunology , Gammacoronavirus/isolation & purification , Immunity, Innate , Interferon-Induced Helicase, IFIH1/genetics , NF-kappa B/genetics , Toll-Like Receptor 7/genetics
13.
Cell Mol Immunol ; 18(4): 945-953, 2021 04.
Article in English | MEDLINE | ID: covidwho-1104474

ABSTRACT

SARS-CoV-2 is the pathogenic agent of COVID-19, which has evolved into a global pandemic. Compared with some other respiratory RNA viruses, SARS-CoV-2 is a poor inducer of type I interferon (IFN). Here, we report that SARS-CoV-2 nsp12, the viral RNA-dependent RNA polymerase (RdRp), suppresses host antiviral responses. SARS-CoV-2 nsp12 attenuated Sendai virus (SeV)- or poly(I:C)-induced IFN-ß promoter activation in a dose-dependent manner. It also inhibited IFN promoter activation triggered by RIG-I, MDA5, MAVS, and IRF3 overexpression. Nsp12 did not impair IRF3 phosphorylation but suppressed the nuclear translocation of IRF3. Mutational analyses suggested that this suppression was not dependent on the polymerase activity of nsp12. Given these findings, our study reveals that SARS-CoV-2 RdRp can antagonize host antiviral innate immunity and thus provides insights into viral pathogenesis.


Subject(s)
COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon Type I/metabolism , SARS-CoV-2/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Nucleus/metabolism , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Type I/genetics , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/metabolism , Interferon-beta/genetics , Interferon-beta/metabolism , Mutation , Phosphorylation , Promoter Regions, Genetic , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , SARS-CoV-2/enzymology , Sendai virus/metabolism
14.
Signal Transduct Target Ther ; 5(1): 299, 2020 12 28.
Article in English | MEDLINE | ID: covidwho-997814

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly spread worldwide and has affected more than 10 million individuals. A typical feature of COVID-19 is the suppression of type I and III interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism by which SARS-CoV-2 evades antiviral immunity remains elusive. Here, we reported that the SARS-CoV-2 membrane (M) protein inhibits the production of type I and III IFNs induced by the cytosolic dsRNA-sensing pathway mediated by RIG-I/MDA-5-MAVS signaling. In addition, the SARS-CoV-2 M protein suppresses type I and III IFN induction stimulated by SeV infection or poly (I:C) transfection. Mechanistically, the SARS-CoV-2 M protein interacts with RIG-I, MAVS, and TBK1, thus preventing the formation of the multiprotein complex containing RIG-I, MAVS, TRAF3, and TBK1 and subsequently impeding the phosphorylation, nuclear translocation, and activation of IRF3. Consequently, ectopic expression of the SARS-CoV-2 M protein facilitates the replication of vesicular stomatitis virus. Taken together, these results indicate that the SARS-CoV-2 M protein antagonizes type I and III IFN production by targeting RIG-I/MDA-5 signaling, which subsequently attenuates antiviral immunity and enhances viral replication. This study provides insight into the interpretation of SARS-CoV-2-induced antiviral immune suppression and illuminates the pathogenic mechanism of COVID-19.


Subject(s)
COVID-19/metabolism , DEAD Box Protein 58/metabolism , Interferon Type I/biosynthesis , Interferon-Induced Helicase, IFIH1/metabolism , Interferons/biosynthesis , SARS-CoV-2/metabolism , Signal Transduction , Viral Matrix Proteins/metabolism , Animals , COVID-19/genetics , Chlorocebus aethiops , DEAD Box Protein 58/genetics , HEK293 Cells , HeLa Cells , Humans , Interferon Type I/genetics , Interferon-Induced Helicase, IFIH1/genetics , Interferons/genetics , Receptors, Immunologic , SARS-CoV-2/genetics , Vero Cells , Viral Matrix Proteins/genetics
15.
Immunogenetics ; 72(6-7): 387-391, 2020 09.
Article in English | MEDLINE | ID: covidwho-690803

ABSTRACT

Covid-19 has caused worldwide devastation. IFIH1 is a pattern recognition receptor that senses coronavirus RNA and triggers interferon production as a first line of viral immune defense. The role of IFIH1 polymorphism, rs1990760 (C>T; aaA946T) in the epidemiology of viral infection is well studied, and the minor allele T resists viral infection. Knock-in mice with mutated IFIH1 protein (946T) for this allele have enhanced interferon production and protection from lethal viral infection. The minor allele frequency (Tmaf) varies widely from Africans (0.06 to 0.35) to Chinese (0.19 to 0.23) to Caucasians (0.56 to 0.69). During the initial days of infection when the social restrictions were not imposed, I show that the infection rate in Italy was lower as expected from its higher Tmaf (0.56) than that in China (Tmaf for southern China, 0.23). The infection rate in the USA and Spain was intermediate between those two countries despite higher Caucasian overall Tmaf (0.69), perhaps due to a more admixed African population in these countries. These analyses suggest that African-Americans and Chinese with low Tmaf of rs1990760 are more vulnerable to SARS-COV2 infection, apart from other genetic factors or socioeconomic conditions in these population. Taken together, an IFN-beta supplement might aid in preventing COVID-19 infection and help in development of herd immunity.


Subject(s)
African Americans/genetics , Coronavirus Infections/genetics , Genetic Predisposition to Disease , Interferon-Induced Helicase, IFIH1/genetics , Pneumonia, Viral/genetics , Betacoronavirus , COVID-19 , China , Gene Frequency , Genotype , Humans , Interferon-beta , Italy , Pandemics , Polymorphism, Single Nucleotide , SARS-CoV-2 , United States
16.
Front Immunol ; 11: 939, 2020.
Article in English | MEDLINE | ID: covidwho-380929

ABSTRACT

Zoonotic infections are an imminent threat to human health. Pangolins were recently identified as carriers and intermediate hosts of coronaviruses. Previous research has shown that infection with coronaviruses activates an innate immune response upon sensing of viral RNA by interferon-induced with helicase C domain 1 (IFIH1), also known as MDA5. Here, we performed a comparative genomics study of RNA sensor genes in three species of pangolins. DDX58/RIG-I, a sensor of cytoplasmic viral RNA and toll-like receptors (TLR) 3, 7, and 8, which bind RNA in endosomes, are conserved in pangolins. By contrast, IFIH1 a sensor of intracellular double-stranded RNA, has been inactivated by mutations in pangolins. Likewise, Z-DNA-binding protein (ZBP1), which senses both Z-DNA and Z-RNA, has been lost during the evolution of pangolins. These results suggest that the innate immune response to viruses differs significantly between pangolins and other mammals, including humans. We put forward the hypothesis that loss of IFIH1 and ZBP1 provided an evolutionary advantage by reducing inflammation-induced damage to host tissues and thereby contributed to a switch from resistance to tolerance of viral infections in pangolins.


Subject(s)
Coronavirus Infections/immunology , Eutheria/virology , Immunity, Innate/genetics , Interferon-Induced Helicase, IFIH1/genetics , Animals , Coronavirus/immunology , DEAD Box Protein 58/genetics , Gene Deletion , Humans , Immunity, Innate/immunology , RNA, Viral/immunology , RNA-Binding Proteins/genetics , Zoonoses/virology
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