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1.
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
2.
Nat Struct Mol Biol ; 28(9): 747-754, 2021 09.
Article in English | MEDLINE | ID: covidwho-1370728

ABSTRACT

Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9 Å resolution cryo-EM structure of the FSE (88 nucleotides, ~28 kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100 nM concentration.


Subject(s)
COVID-19/prevention & control , Cryoelectron Microscopy/methods , Frameshift Mutation/genetics , Oligonucleotides, Antisense/genetics , RNA, Viral/genetics , Response Elements/genetics , SARS-CoV-2/genetics , A549 Cells , Animals , Base Sequence , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Genome, Viral/genetics , Humans , Models, Molecular , Nucleic Acid Conformation , Oligonucleotides, Antisense/pharmacology , RNA, Viral/chemistry , RNA, Viral/ultrastructure , SARS-CoV-2/physiology , SARS-CoV-2/ultrastructure , Vero Cells , Virus Replication/drug effects , Virus Replication/genetics
3.
Viruses ; 13(6)2021 05 21.
Article in English | MEDLINE | ID: covidwho-1359299

ABSTRACT

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress.


Subject(s)
Host-Pathogen Interactions , RNA Viruses/physiology , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Response Elements , Biological Transport , Cytoplasmic Granules/metabolism , Gene Expression Regulation, Viral , Humans , Protein Biosynthesis , RNA Virus Infections/metabolism , RNA Virus Infections/virology , RNA, Viral/chemistry , Stress, Physiological , Transport Vesicles/metabolism , Virus Replication
4.
Cell Chem Biol ; 29(2): 239-248.e4, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1347527

ABSTRACT

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease-associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. Here, we report that TREM2 is a thyroid hormone-regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone and synthetic thyroid hormone agonists (thyromimetics). Our findings report the endocrine regulation of TREM2 by thyroid hormone, and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small-molecule therapeutic agents.


Subject(s)
Acetates/pharmacology , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Membrane Glycoproteins/genetics , Microglia/drug effects , Phenols/pharmacology , Receptors, Immunologic/genetics , Retinoid X Receptors/genetics , Thyroid Hormones/pharmacology , Acetates/chemical synthesis , Animals , Binding Sites , Brain/drug effects , Brain/immunology , Brain/pathology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Gene Expression Regulation , Humans , Immunity, Innate , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C57BL , Microglia/immunology , Microglia/pathology , Models, Molecular , Phenols/chemical synthesis , Phenoxyacetates/pharmacology , Promoter Regions, Genetic , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/immunology , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/immunology , Response Elements , Retinoid X Receptors/chemistry , Retinoid X Receptors/metabolism , Signal Transduction
5.
Mol Cell ; 81(3): 584-598.e5, 2021 02 04.
Article in English | MEDLINE | ID: covidwho-1002917

ABSTRACT

Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as much as 97% of its 30 kilobases have not been structurally explored. Here, we apply a novel long amplicon strategy to determine the secondary structure of the SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells. Our in-depth structural analysis reveals networks of well-folded RNA structures throughout Orf1ab and reveals aspects of SARS-CoV-2 genome architecture that distinguish it from other RNA viruses. Evolutionary analysis shows that several features of the SARS-CoV-2 genomic structure are conserved across ß-coronaviruses, and we pinpoint regions of well-folded RNA structure that merit downstream functional analysis. The native, secondary structure of SARS-CoV-2 presented here is a roadmap that will facilitate focused studies on the viral life cycle, facilitate primer design, and guide the identification of RNA drug targets against COVID-19.


Subject(s)
COVID-19 , Genome, Viral , Nucleic Acid Conformation , RNA, Viral , Response Elements , SARS-CoV-2 , COVID-19/genetics , COVID-19/metabolism , Cell Line, Tumor , Humans , RNA, Viral/genetics , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
6.
Am J Physiol Heart Circ Physiol ; 320(1): H296-H304, 2021 01 01.
Article in English | MEDLINE | ID: covidwho-961166

ABSTRACT

Biological sex is increasingly recognized as a critical determinant of health and disease, particularly relevant to the topical COVID-19 pandemic caused by the SARS-CoV-2 coronavirus. Epidemiological data and observational reports from both the original SARS epidemic and the most recent COVID-19 pandemic have a common feature: males are more likely to exhibit enhanced disease severity and mortality than females. Sex differences in cardiovascular disease and COVID-19 share mechanistic foundations, namely, the involvement of both the innate immune system and the canonical renin-angiotensin system (RAS). Immunological differences suggest that females mount a rapid and aggressive innate immune response, and the attenuated antiviral response in males may confer enhanced susceptibility to severe disease. Furthermore, the angiotensin-converting enzyme 2 (ACE2) is involved in disease pathogenesis in cardiovascular disease and COVID-19, either to serve as a protective mechanism by deactivating the RAS or as the receptor for viral entry, respectively. Loss of membrane ACE2 and a corresponding increase in plasma ACE2 are associated with worsened cardiovascular disease outcomes, a mechanism attributed to a disintegrin and metalloproteinase (ADAM17). SARS-CoV-2 infection also leads to ADAM17 activation, a positive feedback cycle that exacerbates ACE2 loss. Therefore, the relationship between cardiovascular disease and COVID-19 is critically dependent on the loss of membrane ACE2 by ADAM17-mediated proteolytic cleavage. This article explores potential mechanisms involved in COVID-19 that may contribute to sex-specific susceptibility focusing on the innate immune system and the RAS, namely, genetics and sex hormones. Finally, we highlight here the added challenges of gender in the COVID-19 pandemic.


Subject(s)
Adaptive Immunity/immunology , Androgens/immunology , Angiotensin-Converting Enzyme 2/genetics , COVID-19/immunology , Estrogens/immunology , Immunity, Innate/immunology , Receptors, Coronavirus/genetics , ADAM17 Protein/metabolism , Adaptive Immunity/genetics , Androgens/metabolism , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/metabolism , COVID-19/mortality , Cardiovascular Diseases/genetics , Cardiovascular Diseases/immunology , Estrogens/metabolism , Female , Genes, X-Linked/genetics , Genes, X-Linked/immunology , Humans , Immunity, Innate/genetics , Male , Promoter Regions, Genetic , Receptors, Coronavirus/metabolism , Renin-Angiotensin System/genetics , Renin-Angiotensin System/immunology , Response Elements/genetics , SARS-CoV-2/metabolism , Severity of Illness Index , Sex Characteristics , Sex Factors , X Chromosome Inactivation
7.
Sci Rep ; 10(1): 16615, 2020 10 06.
Article in English | MEDLINE | ID: covidwho-834915

ABSTRACT

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Subject(s)
Antibodies, Viral/analysis , Antibody-Dependent Cell Cytotoxicity/immunology , Coronavirus Infections/immunology , Cytotoxicity Tests, Immunologic/methods , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/virology , Genes, Reporter , HEK293 Cells , Humans , Jurkat Cells , Luciferases/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , NFATC Transcription Factors/genetics , Receptors, IgG/genetics , Receptors, IgG/immunology , Response Elements , Spike Glycoprotein, Coronavirus/metabolism , Transfection
8.
Virus Res ; 286: 198074, 2020 09.
Article in English | MEDLINE | ID: covidwho-611212

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Subject(s)
Betacoronavirus/genetics , Host-Pathogen Interactions/genetics , Interferon-beta/genetics , NF-kappa B/genetics , Nucleocapsid Proteins/genetics , Viral Proteins/genetics , Betacoronavirus/immunology , Coronavirus Nucleocapsid Proteins , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Interferon-beta/immunology , Luciferases/genetics , Luciferases/metabolism , NF-kappa B/immunology , Nucleocapsid Proteins/immunology , Phosphoproteins , Plasmids/chemistry , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Response Elements , SARS-CoV-2 , Sendai virus/genetics , Sendai virus/immunology , Signal Transduction , Transfection/methods , Viral Proteins/immunology
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