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1.
Iran J Allergy Asthma Immunol ; 21(2): 219-227, 2022 Apr 11.
Article in English | MEDLINE | ID: covidwho-1818866

ABSTRACT

LPS-responsive beige-like anchor protein (LRBA) deficiency is a primary immunodeficiency caused by a mutation in the LRBA gene. Affected individuals present with a variety of clinical symptoms including hypogammaglobulinemia, recurrent infections, splenomegaly, hepatomegaly, and autoimmune cytopenias. Except for hypogammaglobulinemia, the remaining features resemble autoimmune lymphoproliferative syndrome (ALPS). Here, we report the case of a 14-year-old boy with the ALPS phenotype, eventually diagnosed with LRBA deficiency. He presented with lymphadenopathy and hepatosplenomegaly, along with autoimmune cytopenia. Due to recurrent infections and worsening gastrointestinal symptoms, whole-exome sequencing was conducted and revealed a novel homozygous pathogenic variant in the LRBA gene (c.534del; p.9Asp179IIef*16). The patient recently suffered from clinical deterioration due to SARS-COV-2 which appears to have triggered an acute worsening of his existing Cytomegalovirus colitis leading to an eventual demise. A literature search for reported LRBA deficient patients with ALPS-like phenotype revealed 11 patients. The most common clinical presentations in LRBA patients with ALPS-like phenotype included autoimmunity (100%), splenomegaly (91%), lymphadenopathy (36.4%), and respiratory tract infections (63.6%). LRBA deficiency is unique in the fact that it encompasses immune deficiency, autoimmunity, and lymphoproliferation. In children with multiple symptoms related to these domains, a genetic diagnosis is necessary to ensure tailored and precise medical therapy.


Subject(s)
Agammaglobulinemia , Autoimmune Lymphoproliferative Syndrome , COVID-19 , Common Variable Immunodeficiency , Lymphadenopathy , Protein Deficiency , Adaptor Proteins, Signal Transducing/genetics , Autoimmune Lymphoproliferative Syndrome/diagnosis , Autoimmune Lymphoproliferative Syndrome/genetics , Humans , Lipopolysaccharides , Lymphadenopathy/diagnosis , Male , Phenotype , Reinfection , SARS-CoV-2 , Splenomegaly
2.
Epigenetics Chromatin ; 14(1): 54, 2021 12 11.
Article in English | MEDLINE | ID: covidwho-1571923

ABSTRACT

BACKGROUND: Understanding the molecular basis of susceptibility factors to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global health imperative. It is well-established that males are more likely to acquire SARS-CoV-2 infection and exhibit more severe outcomes. Similarly, exposure to air pollutants and pre-existing respiratory chronic conditions, such as asthma and chronic obstructive respiratory disease (COPD) confer an increased risk to coronavirus disease 2019 (COVID-19). METHODS: We investigated molecular patterns associated with risk factors in 398 candidate genes relevant to COVID-19 biology. To accomplish this, we downloaded DNA methylation and gene expression data sets from publicly available repositories (GEO and GTEx Portal) and utilized data from an empirical controlled human exposure study conducted by our team. RESULTS: First, we observed sex-biased DNA methylation patterns in autosomal immune genes, such as NLRP2, TLE1, GPX1, and ARRB2 (FDR < 0.05, magnitude of DNA methylation difference Δß > 0.05). Second, our analysis on the X-linked genes identified sex associated DNA methylation profiles in genes, such as ACE2, CA5B, and HS6ST2 (FDR < 0.05, Δß > 0.05). These associations were observed across multiple respiratory tissues (lung, nasal epithelia, airway epithelia, and bronchoalveolar lavage) and in whole blood. Some of these genes, such as NLRP2 and CA5B, also exhibited sex-biased gene expression patterns. In addition, we found differential DNA methylation patterns by COVID-19 status for genes, such as NLRP2 and ACE2 in an exploratory analysis of an empirical data set reporting on human COVID-9 infections. Third, we identified modest DNA methylation changes in CpGs associated with PRIM2 and TATDN1 (FDR < 0.1, Δß > 0.05) in response to particle-depleted diesel exhaust in bronchoalveolar lavage. Finally, we captured a DNA methylation signature associated with COPD diagnosis in a gene involved in nicotine dependence (COMT) (FDR < 0.1, Δß > 0.05). CONCLUSION: Our findings on sex differences might be of clinical relevance given that they revealed molecular associations of sex-biased differences in COVID-19. Specifically, our results hinted at a potentially exaggerated immune response in males linked to autosomal genes, such as NLRP2. In contrast, our findings at X-linked loci such as ACE2 suggested a potentially distinct DNA methylation pattern in females that may interact with its mRNA expression and inactivation status. We also found tissue-specific DNA methylation differences in response to particulate exposure potentially capturing a nitrogen dioxide (NO2) effect-a contributor to COVID-19 susceptibility. While we identified a molecular signature associated with COPD, all COPD-affected individuals were smokers, which may either reflect an association with the disease, smoking, or may highlight a compounded effect of these two risk factors in COVID-19. Overall, our findings point towards a molecular basis of variation in susceptibility factors that may partly explain disparities in the risk for SARS-CoV-2 infection.


Subject(s)
COVID-19/genetics , DNA Methylation , Gene Expression , SARS-CoV-2 , Sex Characteristics , Adaptor Proteins, Signal Transducing/genetics , Adolescent , Adult , Air Pollutants/adverse effects , Angiotensin-Converting Enzyme 2/genetics , Apoptosis Regulatory Proteins/genetics , COVID-19/virology , Child , Child, Preschool , Chromosomes, Human, X , Co-Repressor Proteins/genetics , Female , Genes, X-Linked , Glutathione Peroxidase/genetics , Humans , Infant , Infant, Newborn , Male , Middle Aged , Risk Factors , Smoking/adverse effects , Sulfotransferases/genetics , Young Adult , beta-Arrestin 2/genetics
3.
Front Immunol ; 12: 750969, 2021.
Article in English | MEDLINE | ID: covidwho-1551506

ABSTRACT

The COVID-19 is an infectious disease caused by SARS-CoV-2 infection. A large number of clinical studies found high-level expression of pro-inflammatory cytokines in patients infected with SARS-CoV-2, which fuels the rapid development of the disease. However, the specific molecular mechanism is still unclear. In this study, we found that SARS-CoV-2 Nsp5 can induce the expression of cytokines IL-1ß, IL-6, TNF-α, and IL-2 in Calu-3 and THP1 cells. Further research found that Nsp5 enhances cytokine expression through activating the NF-κB signaling pathway. Subsequently, we investigated the upstream effectors of the NF-κB signal pathway on Nsp5 overexpression and discovered that Nsp5 increases the protein level of MAVS. Moreover, Nsp5 can promote the SUMOylation of MAVS to increase its stability and lead to increasing levels of MAVS protein, finally triggering activation of NF-κB signaling. The knockdown of MAVS and the inhibitor of SUMOylation treatment can attenuate Nsp5-mediated NF-κB activation and cytokine induction. We identified a novel role of SARS-CoV-2 Nsp5 to enhance cytokine production by activating the NF-κB signaling pathway.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coronavirus 3C Proteases/immunology , Cytokines/biosynthesis , NF-kappa B/metabolism , SARS-CoV-2/immunology , Sumoylation/physiology , Adaptor Proteins, Signal Transducing/genetics , Animals , COVID-19/immunology , Cell Line , Chlorocebus aethiops , Enzyme Activation/drug effects , HEK293 Cells , Humans , Immunity, Innate/immunology , Interleukin-1beta/biosynthesis , Interleukin-2/biosynthesis , Interleukin-6/biosynthesis , Signal Transduction/physiology , Sumoylation/drug effects , THP-1 Cells , Tumor Necrosis Factor-alpha/biosynthesis , Vero Cells
4.
Nat Commun ; 12(1): 4502, 2021 07 23.
Article in English | MEDLINE | ID: covidwho-1550282

ABSTRACT

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane/metabolism , Cell Nucleus/metabolism , Membrane Glycoproteins/metabolism , Transcription Factors/metabolism , Transcription, Genetic/genetics , Viral Envelope Proteins/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Biological Transport , Cell Fusion , Cell Line , Cell Line, Tumor , Cells, Cultured , Giant Cells/metabolism , HEK293 Cells , Humans , Membrane Glycoproteins/genetics , Mice , RNA-Seq/methods , Signal Transduction/genetics , Transcription Factors/genetics , Viral Envelope Proteins/genetics
5.
Immunity ; 54(12): 2877-2892.e7, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1499988

ABSTRACT

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.


Subject(s)
B-Lymphocytes/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Germinal Center/immunology , SARS-CoV-2/physiology , T-Lymphocytes, Helper-Inducer/immunology , /immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adjuvants, Immunologic , Animals , HEK293 Cells , Humans , Immunity, Humoral , Interleukin-6/genetics , Interleukin-6/metabolism , Liposomes/administration & dosage , Mice , Mice, Inbred BALB C , Nanoparticles/administration & dosage , Protein Subunits/genetics , /genetics
6.
mBio ; 12(5): e0234221, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1494971

ABSTRACT

The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.


Subject(s)
Acyltransferases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Alphacoronavirus/pathogenicity , Nerve Tissue Proteins/metabolism , Acyltransferases/genetics , Adaptor Proteins, Signal Transducing/genetics , Alphacoronavirus/drug effects , Animals , COVID-19/metabolism , HeLa Cells , Humans , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Nerve Tissue Proteins/genetics , Palmitates/pharmacology , SARS Virus/drug effects , SARS Virus/pathogenicity , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Swine
7.
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
9.
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
10.
Signal Transduct Target Ther ; 5(1): 221, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-1387195
11.
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
12.
Adv Sci (Weinh) ; 8(15): e2100606, 2021 08.
Article in English | MEDLINE | ID: covidwho-1340232

ABSTRACT

Mitochondrial antiviral signaling (MAVS) protein is the core signaling adaptor in the RNA signaling pathway. Thus, appropriate regulation of MAVS expression is essential for antiviral immunity against RNA virus infection. However, the regulation of MAVS expression at the mRNA level especially at the post transcriptional level is not well-defined. Here, it is reported that the MAVS mRNA undergoes N6 -methyladenosine (m6 A) modification through methyltransferase-like protein 14 (METTL14), which leads to a fast turnover of MAVS mRNA. Knockdown or deficiency of METTL14 increases MAVS mRNA stability, and downstream phosphorylation of TBK1/IRF3 and interferon-ß production in response to RNA viruses. Compared to wild-type mice, heterozygotes Mettl14+/- mice better tolerate RNA virus infection. The authors' findings unveil a novel mechanism to regulate the stability of MAVS transcripts post-transcriptionally through m6 A modification.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/metabolism , Adenosine/analogs & derivatives , Methyltransferases/immunology , Methyltransferases/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adenosine/genetics , Adenosine/immunology , Adenosine/metabolism , Animals , Disease Models, Animal , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Methyltransferases/genetics , Mice , Mice, Inbred C57BL , Signal Transduction/genetics , Signal Transduction/immunology
13.
Front Immunol ; 12: 683800, 2021.
Article in English | MEDLINE | ID: covidwho-1305645

ABSTRACT

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , Cytokine Release Syndrome/immunology , Interleukin-1 Receptor-Associated Kinases/metabolism , Macrophages/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Gene Expression Regulation , Humans , Immunity, Innate , Inflammation Mediators/metabolism , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-6/genetics , Interleukin-6/metabolism , Lipopolysaccharides/immunology , Macrophages/virology , Protein Binding , THP-1 Cells , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
15.
PLoS One ; 16(6): e0253458, 2021.
Article in English | MEDLINE | ID: covidwho-1286869

ABSTRACT

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC, dACE2 (r≤-0.7, p<0.001) and EPO (r≤-0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , Dopa Decarboxylase/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Angiotensin-Converting Enzyme 2/genetics , Area Under Curve , Aromatic-L-Amino-Acid Decarboxylases , COVID-19/virology , Dopa Decarboxylase/genetics , Down-Regulation , Epithelial Cells/cytology , Epithelial Cells/metabolism , Erythropoietin/genetics , Erythropoietin/metabolism , Female , Humans , Male , Middle Aged , Nasopharynx/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , ROC Curve , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Up-Regulation , Viral Load
16.
PLoS One ; 16(6): e0253089, 2021.
Article in English | MEDLINE | ID: covidwho-1282298

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , COVID-19/immunology , Cell Nucleus/immunology , Interferon Regulatory Factor-3/immunology , RNA-Binding Proteins/immunology , SARS-CoV-2/immunology , Signal Transduction/immunology , Viral Nonstructural Proteins/immunology , Active Transport, Cell Nucleus/genetics , Active Transport, Cell Nucleus/immunology , Adaptor Proteins, Signal Transducing/genetics , COVID-19/genetics , Cell Nucleus/genetics , HeLa Cells , Humans , Interferon Regulatory Factor-3/genetics , NF-kappa B/genetics , NF-kappa B/immunology , Phosphorylation/genetics , Phosphorylation/immunology , /immunology , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Signal Transduction/genetics , Viral Nonstructural Proteins/genetics
17.
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
18.
Nutrients ; 13(5)2021 May 10.
Article in English | MEDLINE | ID: covidwho-1224082

ABSTRACT

BACKGROUND: Acute and chronic alcohol abuse has adverse impacts on both the innate and adaptive immune response, which may result in reduced resistance to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and promote the progression of coronavirus disease 2019 (COVID-19). However, there are no large population-based data evaluating potential causal associations between alcohol consumption and COVID-19. METHODS: We conducted a Mendelian randomization study using data from UK Biobank to explore the association between alcohol consumption and risk of SARS-CoV-2 infection and serious clinical outcomes in patients with COVID-19. A total of 12,937 participants aged 50-83 who tested for SARS-CoV-2 between 16 March to 27 July 2020 (12.1% tested positive) were included in the analysis. The exposure factor was alcohol consumption. Main outcomes were SARS-CoV-2 positivity and death in COVID-19 patients. We generated allele scores using three genetic variants (rs1229984 (Alcohol Dehydrogenase 1B, ADH1B), rs1260326 (Glucokinase Regulator, GCKR), and rs13107325 (Solute Carrier Family 39 Member 8, SLC39A8)) and applied the allele scores as the instrumental variables to assess the effect of alcohol consumption on outcomes. Analyses were conducted separately for white participants with and without obesity. RESULTS: Of the 12,937 participants, 4496 were never or infrequent drinkers and 8441 were frequent drinkers. Both logistic regression and Mendelian randomization analyses found no evidence that alcohol consumption was associated with risk of SARS-CoV-2 infection in participants either with or without obesity (All q > 0.10). However, frequent drinking, especially heavy drinking (HR = 2.07, 95%CI 1.24-3.47; q = 0.054), was associated with higher risk of death in patients with obesity and COVID-19, but not in patients without obesity. Notably, the risk of death in frequent drinkers with obesity increased slightly with the average amount of alcohol consumed weekly (All q < 0.10). CONCLUSIONS: Our findings suggest that alcohol consumption has adverse effects on the progression of COVID-19 in white participants with obesity, but was not associated with susceptibility to SARS-CoV-2 infection.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Alcohol Dehydrogenase/genetics , Alcohol Drinking , Biological Specimen Banks , COVID-19 , Cation Transport Proteins/genetics , Obesity , Polymorphism, Single Nucleotide , SARS Virus , Aged , Alcohol Drinking/genetics , Alcohol Drinking/mortality , COVID-19/genetics , COVID-19/mortality , Disease-Free Survival , Female , Humans , Male , Mendelian Randomization Analysis , Middle Aged , Obesity/genetics , Obesity/mortality , Survival Rate , United Kingdom/epidemiology
19.
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
20.
J Biol Chem ; 295(51): 17781-17801, 2020 12 18.
Article in English | MEDLINE | ID: covidwho-985572

ABSTRACT

Knockout mouse models have been extensively used to study the antiviral activity of IFIT (interferon-induced protein with tetratricopeptide repeats). Human IFIT1 binds to cap0 (m7GpppN) RNA, which lacks methylation on the first and second cap-proximal nucleotides (cap1, m7GpppNm, and cap2, m7GpppNmNm, respectively). These modifications are signatures of "self" in higher eukaryotes, whereas unmodified cap0-RNA is recognized as foreign and, therefore, potentially harmful to the host cell. IFIT1 inhibits translation at the initiation stage by competing with the cap-binding initiation factor complex, eIF4F, restricting infection by certain viruses that possess "nonself" cap0-mRNAs. However, in mice and other rodents, the IFIT1 orthologue has been lost, and the closely related Ifit1b has been duplicated twice, yielding three paralogues: Ifit1, Ifit1b, and Ifit1c. Although murine Ifit1 is similar to human IFIT1 in its cap0-RNA-binding selectivity, the roles of Ifit1b and Ifit1c are unknown. Here, we found that Ifit1b preferentially binds to cap1-RNA, whereas binding is much weaker to cap0- and cap2-RNA. In murine cells, we show that Ifit1b can modulate host translation and restrict WT mouse coronavirus infection. We found that Ifit1c acts as a stimulatory cofactor for both Ifit1 and Ifit1b, promoting their translation inhibition. In this way, Ifit1c acts in an analogous fashion to human IFIT3, which is a cofactor to human IFIT1. This work clarifies similarities and differences between the human and murine IFIT families to facilitate better design and interpretation of mouse models of human infection and sheds light on the evolutionary plasticity of the IFIT family.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Coronavirus/growth & development , Coronavirus/genetics , Protein Biosynthesis , RNA Cap-Binding Proteins/metabolism , RNA Caps/metabolism , RNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Coronavirus/metabolism , Disease Models, Animal , HEK293 Cells , Humans , Mice , Mice, Knockout , Models, Molecular , Mutation , Protein Binding , RAW 264.7 Cells , RNA-Binding Proteins/genetics
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