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1.
Viruses ; 13(8)2021 08 23.
Article in English | MEDLINE | ID: covidwho-1524167

ABSTRACT

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , SARS-CoV-2/drug effects , Tretinoin/pharmacology , Animals , Cell Line, Tumor , Chlorocebus aethiops , Cysteine Proteinase Inhibitors/pharmacology , DEAD Box Protein 58/metabolism , High-Throughput Screening Assays , Humans , Receptors, Immunologic/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Vero Cells , Virus Replication/drug effects
2.
Curr Issues Mol Biol ; 43(3): 1212-1225, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1438531

ABSTRACT

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. However, the factors predisposing individuals to severe disease remain poorly understood. Here, we show that levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells, are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air-liquid interface cultures of primary human bronchial epithelial cells. Moreover, SARS-CoV-2 infection increases SIRPalpha levels, the binding partner of CD47, on primary human monocytes. Systematic literature searches further indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions that may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, age-related and virus-induced CD47 expression is a candidate mechanism potentially contributing to severe COVID-19, as well as a therapeutic target, which may be addressed by antibodies and small molecules. Further research will be needed to investigate the potential involvement of CD47 and SIRPalpha in COVID-19 pathology. Our data should encourage other research groups to consider the potential relevance of the CD47/ SIRPalpha axis in their COVID-19 research.


Subject(s)
Antigens, Differentiation/metabolism , CD47 Antigen/metabolism , COVID-19/epidemiology , COVID-19/metabolism , Pandemics , Receptors, Immunologic/metabolism , SARS-CoV-2/metabolism , Severity of Illness Index , Signal Transduction/immunology , Blood Donors , Blotting, Western/methods , Bronchi/cytology , COVID-19/pathology , COVID-19/virology , Caco-2 Cells , Epithelial Cells/metabolism , Epithelial Cells/virology , Healthy Volunteers , Humans , Monocytes/metabolism , Monocytes/virology , Polymerase Chain Reaction/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
3.
mBio ; 12(5): e0233521, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1430167

ABSTRACT

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coronavirus 3C Proteases/metabolism , DEAD Box Protein 58/metabolism , Receptors, Immunologic/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , A549 Cells , Adaptor Proteins, Signal Transducing/genetics , Animals , Caco-2 Cells , Coronavirus 3C Proteases/genetics , DEAD Box Protein 58/genetics , Enzyme-Linked Immunosorbent Assay , HCT116 Cells , HEK293 Cells , Humans , Immunity, Innate/genetics , Immunity, Innate/physiology , Immunoblotting , Interferon Type I/metabolism , Mice , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/physiology , Ubiquitination , Virus Replication/genetics , Virus Replication/physiology
4.
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
5.
Cytokine ; 148: 155697, 2021 12.
Article in English | MEDLINE | ID: covidwho-1385382

ABSTRACT

The prevalence of SARS-CoV-2 is a great threat to global public health. However, the relationship between the viral pathogen SARS-CoV-2 and host innate immunity has not yet been well studied. The genome of SARS-CoV-2 encodes a viral protease called 3C-like protease. This protease is responsible for cleaving viral polyproteins during replication. In this investigation, 293T cells were transfected with SARS-CoV-2 3CL and then infected with Sendai virus (SeV) to induce the RIG-I like receptor (RLR)-based immune pathway. q-PCR, luciferase reporter assays, and western blotting were used for experimental analyses. We found that SARS-CoV-2 3CL significantly downregulated IFN-ß mRNA levels. Upon SeV infection, SARS-CoV-2 3CL inhibited the nuclear translocation of IRF3 and p65 and promoted the degradation of IRF3. This effect of SARS-CoV-2 3CL on type I IFN in the RLR immune pathway opens up novel ideas for future research on SARS-CoV-2.


Subject(s)
Coronavirus 3C Proteases/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon-beta/biosynthesis , Proteolysis , DEAD Box Protein 58/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Interferon-beta/genetics , NF-kappa B/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Immunologic/metabolism , Response Elements/genetics , Sendai virus/physiology , Signal Transduction
6.
Cell Signal ; 87: 110121, 2021 11.
Article in English | MEDLINE | ID: covidwho-1370457

ABSTRACT

The SARS-CoV-2 virus has caused a worldwide COVID-19 pandemic. In less than a year and a half, more than 200 million people have been infected and more than four million have died. Despite some improvement in the treatment strategies, no definitive treatment protocol has been developed. The pathogenesis of the disease has not been clearly elucidated yet. A clear understanding of its pathogenesis will help develop effective vaccines and drugs. The immunopathogenesis of COVID-19 is characteristic with acute respiratory distress syndrome and multiorgan involvement with impaired Type I interferon response and hyperinflammation. The destructive systemic effects of COVID-19 cannot be explained simply by the viral tropism through the ACE2 and TMPRSS2 receptors. In addition, the recently identified mutations cannot fully explain the defect in all cases of Type I interferon synthesis. We hypothesize that retinol depletion and resulting impaired retinoid signaling play a central role in the COVID-19 pathogenesis that is characteristic for dysregulated immune system, defect in Type I interferon synthesis, severe inflammatory process, and destructive systemic multiorgan involvement. Viral RNA recognition mechanism through RIG-I receptors can quickly consume a large amount of the body's retinoid reserve, which causes the retinol levels to fall below the normal serum levels. This causes retinoid insufficiency and impaired retinoid signaling, which leads to interruption in Type I interferon synthesis and an excessive inflammation. Therefore, reconstitution of the retinoid signaling may prove to be a valid strategy for management of COVID-19 as well for some other chronic, degenerative, inflammatory, and autoimmune diseases.


Subject(s)
COVID-19/pathology , Signal Transduction/physiology , Vitamin A/metabolism , COVID-19/immunology , COVID-19/metabolism , COVID-19/virology , Central Nervous System/metabolism , DEAD Box Protein 58/metabolism , Humans , Immune Tolerance , Interferon Type I/metabolism , Receptors, Immunologic/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Viral Tropism/physiology , Vitamin A/blood
7.
Int J Mol Sci ; 22(15)2021 Jul 23.
Article in English | MEDLINE | ID: covidwho-1346497

ABSTRACT

Platelets are hematopoietic cells whose main function has for a long time been considered to be the maintenance of vascular integrity. They have an essential role in the hemostatic response, but they also have functional capabilities that go far beyond it. This review will provide an overview of platelet functions. Indeed, stress signals may induce platelet apoptosis through proapoptotis or hemostasis receptors, necrosis, and even autophagy. Platelets also interact with immune cells and modulate immune responses in terms of activation, maturation, recruitment and cytokine secretion. This review will also show that platelets, thanks to their wide range of innate immune receptors, and in particular toll-like receptors, and can be considered sentinels actively participating in the immuno-surveillance of the body. We will discuss the diversity of platelet responses following the engagement of these receptors as well as the signaling pathways involved. Finally, we will show that while platelets contribute significantly, via their TLRs, to immune response and inflammation, these receptors also participate in the pathophysiological processes associated with various pathogens and diseases, including cancer and atherosclerosis.


Subject(s)
Atherosclerosis/pathology , Blood Platelets/pathology , Immunity, Innate/immunology , Neoplasms/pathology , Platelet Activation , Receptors, Immunologic/metabolism , Toll-Like Receptors/metabolism , Animals , Atherosclerosis/immunology , Atherosclerosis/metabolism , Blood Platelets/immunology , Blood Platelets/metabolism , Humans , Neoplasms/immunology , Neoplasms/metabolism
8.
Front Immunol ; 12: 688758, 2021.
Article in English | MEDLINE | ID: covidwho-1304592

ABSTRACT

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/metabolism , Interferons/antagonists & inhibitors , Interferons/immunology , Receptors, Immunologic/metabolism , Amino Acid Sequence/genetics , Animals , COVID-19/pathology , DEAD Box Protein 58/immunology , Deltacoronavirus/genetics , Deltacoronavirus/immunology , Humans , Infectious bronchitis virus/genetics , Infectious bronchitis virus/immunology , Interferon Regulatory Factor-3/metabolism , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Phosphorylation , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/immunology , Receptors, Immunologic/immunology , SARS Virus/genetics , SARS Virus/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Swine , Ubiquitination/physiology
9.
Front Immunol ; 12: 688758, 2021.
Article in English | MEDLINE | ID: covidwho-1295641

ABSTRACT

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.


Subject(s)
Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/immunology , DEAD Box Protein 58/metabolism , Interferons/antagonists & inhibitors , Interferons/immunology , Receptors, Immunologic/metabolism , Amino Acid Sequence/genetics , Animals , COVID-19/pathology , DEAD Box Protein 58/immunology , Deltacoronavirus/genetics , Deltacoronavirus/immunology , Humans , Infectious bronchitis virus/genetics , Infectious bronchitis virus/immunology , Interferon Regulatory Factor-3/metabolism , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Phosphorylation , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/immunology , Receptors, Immunologic/immunology , SARS Virus/genetics , SARS Virus/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Swine , Ubiquitination/physiology
10.
Front Immunol ; 12: 662989, 2021.
Article in English | MEDLINE | ID: covidwho-1256380

ABSTRACT

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of current COVID-19 pandemic, and insufficient production of type I interferon (IFN-I) is associated with the severe forms of the disease. Membrane (M) protein of SARS-CoV-2 has been reported to suppress host IFN-I production, but the underlying mechanism is not completely understood. In this study, SARS-CoV-2 M protein was confirmed to suppress the expression of IFNß and interferon-stimulated genes induced by RIG-I, MDA5, IKKϵ, and TBK1, and to inhibit IRF3 phosphorylation and dimerization caused by TBK1. SARS-CoV-2 M could interact with MDA5, TRAF3, IKKϵ, and TBK1, and induce TBK1 degradation via K48-linked ubiquitination. The reduced TBK1 further impaired the formation of TRAF3-TANK-TBK1-IKKε complex that leads to inhibition of IFN-I production. Our study revealed a novel mechanism of SARS-CoV-2 M for negative regulation of IFN-I production, which would provide deeper insight into the innate immunosuppression and pathogenicity of SARS-CoV-2.


Subject(s)
Interferon Type I/biosynthesis , SARS-CoV-2/immunology , Ubiquitin/metabolism , Viral Matrix Proteins/immunology , DEAD Box Protein 58/metabolism , HEK293 Cells , Humans , I-kappa B Kinase/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon-Induced Helicase, IFIH1/metabolism , Proteolysis , Receptors, Immunologic/metabolism , Signal Transduction , TNF Receptor-Associated Factor 3/metabolism
11.
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
12.
Cell Rep ; 35(6): 109091, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1213072

ABSTRACT

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3' end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.


Subject(s)
COVID-19/genetics , Methyltransferases/metabolism , SARS-CoV-2/genetics , Adenosine/metabolism , COVID-19/metabolism , Cell Line , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , Humans , Immunity, Innate/genetics , Methylation , Methyltransferases/genetics , RNA, Viral/genetics , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , SARS-CoV-2/pathogenicity , Signal Transduction
13.
Front Immunol ; 12: 666983, 2021.
Article in English | MEDLINE | ID: covidwho-1186803

ABSTRACT

The emergence of viruses with pandemic potential such as the SARS-CoV-2 coronavirus causing COVID-19 poses a global health challenge. There is remarkable progress in vaccine technology in response to this threat, but their design often overlooks the innate arm of immunity. Gamma Delta (γδ) T cells are a subset of T cells with unique features that gives them a key role in the innate immune response to a variety of homeostatic alterations, from cancer to microbial infections. In the context of viral infection, a growing body of evidence shows that γδ T cells are particularly equipped for early virus detection, which triggers their subsequent activation, expansion and the fast deployment of antiviral functions such as direct cytotoxic pathways, secretion of cytokines, recruitment and activation of other immune cells and mobilization of a trained immunity memory program. As such, γδ T cells represent an attractive target to stimulate for a rapid and effective resolution of viral infections. Here, we review the known aspects of γδ T cells that make them crucial component of the immune response to viruses, and the ways that their antiviral potential can be harnessed to prevent or treat viral infection.


Subject(s)
COVID-19/immunology , COVID-19/virology , Host-Pathogen Interactions , Receptors, Antigen, T-Cell, gamma-delta/metabolism , SARS-CoV-2/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Adaptive Immunity , Animals , COVID-19/drug therapy , Combined Modality Therapy , Cytotoxicity, Immunologic , Disease Management , Disease Susceptibility/immunology , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Receptors, Immunologic/metabolism
15.
Cells ; 10(3)2021 03 02.
Article in English | MEDLINE | ID: covidwho-1125490

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.


Subject(s)
Coronavirus Nucleocapsid Proteins/metabolism , DEAD Box Protein 58/metabolism , Interferons/metabolism , Receptors, Immunologic/metabolism , SARS-CoV-2/metabolism , DEAD Box Protein 58/genetics , Host-Pathogen Interactions/genetics , Humans , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Interferon Type I/genetics , Interferon Type I/metabolism , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interferons/genetics , Orthomyxoviridae/genetics , Orthomyxoviridae/metabolism , Phosphoproteins/metabolism , Poly C/pharmacology , Poly I/pharmacology , Promoter Regions, Genetic , /metabolism , Receptors, Immunologic/genetics , SARS-CoV-2/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription Factors/metabolism , Tripartite Motif Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Up-Regulation , Zika Virus/genetics , Zika Virus/metabolism
16.
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
17.
J Infect Dis ; 222(12): 1985-1996, 2020 11 13.
Article in English | MEDLINE | ID: covidwho-1059699

ABSTRACT

BACKGROUND: An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease. METHODS: An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis. RESULTS: Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance. CONCLUSIONS: Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.


Subject(s)
COVID-19/immunology , Granulocytes/immunology , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Adult , Aged , Biomarkers/metabolism , CD11b Antigen/immunology , COVID-19/blood , COVID-19/diagnosis , Female , France , Humans , Immunophenotyping , Leukocyte Count , Lymphocytes/immunology , Male , Middle Aged , Monocytes/immunology , Prospective Studies , SARS-CoV-2 , Severity of Illness Index
18.
Immunobiology ; 226(1): 152021, 2021 01.
Article in English | MEDLINE | ID: covidwho-908903

ABSTRACT

SARS-CoV-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. As per the literature, the SARS-CoV-2 is known to exploit humanACE2 receptors (similar toprevious SARS-CoV-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. However, considering the higher infectivity of SARS-CoV-2 along with the complex etiology and pathophysiological outcomes seen in COVID-19 patients, it seems that there may be an alternate receptor for SARS-CoV-2. I performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of SARS-CoV-2 as compared to previous known coronaviruses. My study revealed that SARS-CoV-2 (previously known as 2019-nCoV) harbors a RGD motif in its receptor binding domain (RBD) and the motif is absent in all other previously known SARS-CoVs. The RGD motif is well known for its role in cell-attachment and cell-adhesion. My hypothesis is that the SARS-CoV-2 may be (via RGD) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. However, an experimental verification is required. The expression of ACE2, which is a known receptor for SARS-CoV-2, was found to be negligible in lungs. I assume that higher infectivity of SARS-CoV-2 could be due to this RGD-integrin mediated acquired cell-adhesive property. Gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvß6, α5ß1, αvß8 and an ECM protein, ICAM1. The molecular docking experiment showed the RBD of spike protein binds with integrins precisely at RGD motif in a similar manner as a synthetic RGD peptide binds to integrins as found by other researchers. SARS-CoV-2 spike protein has a number of phosphorylation sites that can induce cAMP, PKC, Tyr signaling pathways. These pathways either activate calcium ion channels or get activated by calcium. In fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of RGD-integrin docking site in our analysis which suggests that RGD-integrins interaction possibly occurs in calcium-dependent manner. The higher expression of integrins in lungs along with their previously known high binding affinity (~KD = 4.0 nM) for virus RGD motif could serve as a possible explanation for high infectivity of SARS-CoV-2. On the contrary, human ACE2 has lower expression in lungs and its high binding affinity (~KD = 15 nM) for spike RBD alone could not manifest significant virus-host attachment. This suggests that besides human ACE2, an additional or alternate receptor for SARS-CoV-2 is likely to exist. A highly relevant evidence never reported earlier which corroborate in favor of RGD-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of TNF-α and IL-6 activation; and integrins role in their activation is also well established. Altogether, the current study has highlighted possible role of calcium and other divalent ions in RGD-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment.


Subject(s)
COVID-19/virology , Calcium/metabolism , Chelation Therapy , Edetic Acid/therapeutic use , Integrins/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites/genetics , COVID-19/drug therapy , Calcium Channels/metabolism , Gene Expression Profiling , Humans , Integrins/chemistry , Intercellular Adhesion Molecule-1/metabolism , Interleukin-6/metabolism , Lung/metabolism , Molecular Docking Simulation , Oligopeptides/chemistry , Oligopeptides/metabolism , Protein Binding , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Sequence Alignment , Signal Transduction/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Tumor Necrosis Factor-alpha/metabolism , Virus Attachment
19.
Nat Immunol ; 21(9): 1107-1118, 2020 09.
Article in English | MEDLINE | ID: covidwho-710376

ABSTRACT

In coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the relationship between disease severity and the host immune response is not fully understood. Here we performed single-cell RNA sequencing in peripheral blood samples of 5 healthy donors and 13 patients with COVID-19, including moderate, severe and convalescent cases. Through determining the transcriptional profiles of immune cells, coupled with assembled T cell receptor and B cell receptor sequences, we analyzed the functional properties of immune cells. Most cell types in patients with COVID-19 showed a strong interferon-α response and an overall acute inflammatory response. Moreover, intensive expansion of highly cytotoxic effector T cell subsets, such as CD4+ effector-GNLY (granulysin), CD8+ effector-GNLY and NKT CD160, was associated with convalescence in moderate patients. In severe patients, the immune landscape featured a deranged interferon response, profound immune exhaustion with skewed T cell receptor repertoire and broad T cell expansion. These findings illustrate the dynamic nature of immune responses during disease progression.


Subject(s)
Antigens, CD/metabolism , Antigens, Differentiation, T-Lymphocyte/metabolism , Betacoronavirus/immunology , Coronavirus Infections/immunology , Interferon Type I/metabolism , Pneumonia, Viral/immunology , Receptors, Immunologic/metabolism , Adolescent , Adult , Aged , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Differentiation, T-Lymphocyte/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , COVID-19 , Cohort Studies , Coronavirus Infections/blood , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Female , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , GPI-Linked Proteins/metabolism , Humans , Interferon Type I/genetics , Interferon Type I/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Middle Aged , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , RNA-Seq , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology , SARS-CoV-2 , Severity of Illness Index , Single-Cell Analysis
20.
Med Hypotheses ; 143: 110122, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-663829

ABSTRACT

A characteristic feature of COVID-19 disease is lymphopenia. Lymphopenia occurs early in the clinical course and is a predictor of disease severity and outcomes. The mechanism of lymphopenia in COVID-19 is uncertain. It has been variously attributed to the release of inflammatory cytokines including IL-6 and TNF-α; direct infection of the lymphocytes by the virus; and rapid sequestration of lymphocytes in the tissues. Additionally, we postulate that prostaglandin D2 (PGD2) is a key meditator of lymphopenia in COVID-19. First, SARS-CoV infection is known to stimulate the production of PGD2 in the airways, which inhibits the host dendritic cell response via the DP1 receptor signaling. Second, PGD2 is known to upregulate monocytic myeloid-derived suppressor cells (MDSC) via the DP2 receptor signaling in group 2 innate lymphoid cells (ILC2). We propose targeting PGD2/DP2 signaling using a receptor antagonist such as ramatroban as an immunotherapy for immune dysfunction and lymphopenia in COVID-19 disease.


Subject(s)
Betacoronavirus , Coronavirus Infections/physiopathology , Lymphopenia/physiopathology , Models, Immunological , Molecular Targeted Therapy , Pandemics , Pneumonia, Viral/physiopathology , Prostaglandin D2/physiology , Respiratory System/metabolism , Adult , COVID-19 , Carbazoles/pharmacology , Carbazoles/therapeutic use , Child , Coronavirus Infections/complications , Coronavirus Infections/immunology , Dendritic Cells/immunology , Humans , Lymphopenia/etiology , Myeloid Cells/immunology , Pneumonia, Viral/complications , Pneumonia, Viral/immunology , Prostaglandin D2/biosynthesis , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/antagonists & inhibitors , Receptors, Prostaglandin/metabolism , Receptors, Prostaglandin/physiology , SARS-CoV-2 , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , T-Lymphocytes/immunology , Thromboxane A2/antagonists & inhibitors
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