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1.
Int J Mol Med ; 49(2)2022 02.
Article in English | MEDLINE | ID: covidwho-1594678

ABSTRACT

The pathophysiology of coronavirus disease 2019 (COVID­19) is mainly dependent on the underlying mechanisms that mediate the entry of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) into the host cells of the various human tissues/organs. Recent studies have indicated a higher order of complexity of the mechanisms of infectivity, given that there is a wide­repertoire of possible cell entry mediators that appear to co­localise in a cell­ and tissue­specific manner. The present study provides an overview of the 'canonical' SARS­CoV­2 mediators, namely angiotensin converting enzyme 2, transmembrane protease serine 2 and 4, and neuropilin­1, expanding on the involvement of novel candidates, including glucose­regulated protein 78, basigin, kidney injury molecule­1, metabotropic glutamate receptor subtype 2, ADAM metallopeptidase domain 17 (also termed tumour necrosis factor­α convertase) and Toll­like receptor 4. Furthermore, emerging data indicate that changes in microRNA (miRNA/miR) expression levels in patients with COVID­19 are suggestive of further complexity in the regulation of these viral mediators. An in silico analysis revealed 160 candidate miRNAs with potential strong binding capacity in the aforementioned genes. Future studies should concentrate on elucidating the association between the cellular tropism of the SARS­CoV­2 cell entry mediators and the mechanisms through which they might affect the clinical outcome. Finally, the clinical utility as a biomarker or therapeutic target of miRNAs in the context of COVID­19 warrants further investigation.


Subject(s)
COVID-19/metabolism , MicroRNAs/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Virus Internalization , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/virology , /metabolism , Gene Expression Regulation , Host-Pathogen Interactions , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , MicroRNAs/genetics , Neuropilin-1/genetics , Neuropilin-1/metabolism , Receptors, Virus/genetics , SARS-CoV-2/physiology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Viral Tropism
2.
Immunity ; 54(11): 2632-2649.e6, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1549842

ABSTRACT

The incidence and severity of sepsis is higher among individuals of African versus European ancestry. We found that genetic risk variants (RVs) in the trypanolytic factor apolipoprotein L1 (APOL1), present only in individuals of African ancestry, were associated with increased sepsis incidence and severity. Serum APOL1 levels correlated with sepsis and COVID-19 severity, and single-cell sequencing in human kidneys revealed high expression of APOL1 in endothelial cells. Analysis of mice with endothelial-specific expression of RV APOL1 and in vitro studies demonstrated that RV APOL1 interfered with mitophagy, leading to cytosolic release of mitochondrial DNA and activation of the inflammasome (NLRP3) and the cytosolic nucleotide sensing pathways (STING). Genetic deletion or pharmacological inhibition of NLRP3 and STING protected mice from RV APOL1-induced permeability defects and proinflammatory endothelial changes in sepsis. Our studies identify the inflammasome and STING pathways as potential targets to reduce APOL1-associated health disparities in sepsis and COVID-19.


Subject(s)
Apolipoprotein L1/genetics , COVID-19/genetics , Genetic Predisposition to Disease/genetics , Sepsis/genetics , Animals , Apolipoprotein L1/blood , COVID-19/pathology , DNA, Mitochondrial/metabolism , Endothelial Cells/metabolism , Humans , Inflammation/genetics , Inflammation/pathology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Mitophagy/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Risk Factors , Sepsis/pathology , Severity of Illness Index , /statistics & numerical data
3.
Front Endocrinol (Lausanne) ; 12: 714909, 2021.
Article in English | MEDLINE | ID: covidwho-1497067

ABSTRACT

Background: Clinically, evidence shows that uterine corpus endometrial carcinoma (UCEC) patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may have a higher death-rate. However, current anti-UCEC/coronavirus disease 2019 (COVID-19) treatment is lacking. Plumbagin (PLB), a pharmacologically active alkaloid, is an emerging anti-cancer inhibitor. Accordingly, the current report was designed to identify and characterize the anti-UCEC function and mechanism of PLB in the treatment of patients infected with SARS-CoV-2 via integrated in silico analysis. Methods: The clinical analyses of UCEC and COVID-19 in patients were conducted using online-accessible tools. Meanwhile, in silico methods including network pharmacology and biological molecular docking aimed to screen and characterize the anti-UCEC/COVID-19 functions, bio targets, and mechanisms of the action of PLB. Results: The bioinformatics data uncovered the clinical characteristics of UCEC patients infected with SARS-CoV-2, including specific genes, health risk, survival rate, and prognostic index. Network pharmacology findings disclosed that PLB-exerted anti-UCEC/COVID-19 effects were achieved through anti-proliferation, inducing cytotoxicity and apoptosis, anti-inflammation, immunomodulation, and modulation of some of the key molecular pathways associated with anti-inflammatory and immunomodulating actions. Following molecular docking analysis, in silico investigation helped identify the anti-UCEC/COVID-19 pharmacological bio targets of PLB, including mitogen-activated protein kinase 3 (MAPK3), tumor necrosis factor (TNF), and urokinase-type plasminogen activator (PLAU). Conclusions: Based on the present bioinformatic and in silico findings, the clinical characterization of UCEC/COVID-19 patients was revealed. The candidate, core bio targets, and molecular pathways of PLB action in the potential treatment of UCEC/COVID-19 were identified accordingly.


Subject(s)
COVID-19 , Carcinoma, Endometrioid , Endometrial Neoplasms , Host-Pathogen Interactions , Naphthoquinones/pharmacology , Adult , Aged , Aged, 80 and over , COVID-19/complications , COVID-19/diagnosis , COVID-19/drug therapy , COVID-19/genetics , Calcium-Binding Proteins/drug effects , Calcium-Binding Proteins/metabolism , Carcinoma, Endometrioid/complications , Carcinoma, Endometrioid/diagnosis , Carcinoma, Endometrioid/drug therapy , Carcinoma, Endometrioid/genetics , Computational Biology , Drug Screening Assays, Antitumor/methods , Endometrial Neoplasms/complications , Endometrial Neoplasms/diagnosis , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Regulatory Networks/drug effects , Genetic Association Studies , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Middle Aged , Mitogen-Activated Protein Kinase 3/drug effects , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Docking Simulation/methods , Naphthoquinones/therapeutic use , Prognosis , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Signal Transduction/drug effects , Signal Transduction/genetics , Tumor Necrosis Factor-alpha/drug effects , Tumor Necrosis Factor-alpha/metabolism , Uterus/drug effects , Uterus/metabolism , Uterus/pathology , Uterus/virology
4.
Biol Direct ; 16(1): 20, 2021 10 21.
Article in English | MEDLINE | ID: covidwho-1477450

ABSTRACT

SARS-CoV-2 infection could cause severe acute respiratory syndrome, largely attributed to dysregulated immune activation and extensive lung tissue damage. However, the underlying mechanisms are not fully understood. Here, we reported that viral infection could induce syncytia formation within cells expressing ACE2 and the SARS-CoV-2 spike protein, leading to the production of micronuclei with an average rate of about 4 per syncytium (> 93%). Remarkably, these micronuclei were manifested with a high level of activation of both DNA damage response and cGAS-STING signaling, as indicated by micronucleus translocation of γH2Ax and cGAS, and upregulation of their respective downstream target genes. Since activation of these signaling pathways were known to be associated with cellular catastrophe and aberrant immune activation, these findings help explain the pathological effects of SARS-CoV-2 infection at cellular and molecular levels, and provide novel potential targets for COVID-19 therapy.


Subject(s)
COVID-19/genetics , COVID-19/metabolism , DNA Damage , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/virology , Giant Cells/metabolism , Giant Cells/virology , HeLa Cells , Humans , Micronucleus Tests , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
5.
ACS Chem Biol ; 16(5): 844-856, 2021 05 21.
Article in English | MEDLINE | ID: covidwho-1457790

ABSTRACT

Interferon-induced transmembrane proteins (IFITMs) are S-palmitoylated proteins in vertebrates that restrict a diverse range of viruses. S-palmitoylated IFITM3 in particular engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, how S-palmitoylation modulates the structure and biophysical characteristics of IFITM3 to promote its antiviral activity remains unclear. To investigate how site-specific S-palmitoylation controls IFITM3 antiviral activity, we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of cysteine 72 enhances the antiviral activity of IFITM3 by modulating its conformation and interaction with lipid membranes. Collectively, our results demonstrate that site-specific S-palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.


Subject(s)
Antiviral Agents/chemistry , Cell Membrane/metabolism , Interferons/chemistry , Lipids/chemistry , Membrane Proteins/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Antiviral Agents/pharmacology , Binding Sites , Cell Membrane/ultrastructure , Computational Biology , Drug Design , Humans , Interferons/pharmacology , Lipoylation , Lysosomes/metabolism , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Signal Transduction
6.
Microbiol Spectr ; 9(2): e0119921, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1398600

ABSTRACT

Human angiotensin I-converting enzyme 2 (hACE2) is a type I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for the attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment, or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 was localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV or SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation does not play a role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell-to-cell fusion mediated by SARS-CoV S protein but not that mediated by SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which may be the result of low cell surface expression of the deglycosylated ACE2 receptor. IMPORTANCE Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction. In addition, we found that the removal of N-glycans of ACE2 impaired its ability to support an efficient transduction of SARS-CoV and SARS-CoV-2 S pseudotyped viruses. Our data suggest that the role of deglycosylation of ACE2 on reducing infection is likely due to a reduced expression of the viral receptor on the cell surface. These findings offer insight into the glycan structure and function of ACE2 and potentially suggest that future antiviral therapies against coronaviruses and other coronavirus-related illnesses involving inhibition of ACE2 recruitment to the cell membrane could be developed.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/growth & development , Tunicamycin/pharmacology , Virus Attachment/drug effects , Virus Internalization/drug effects , Animals , Antiviral Agents/pharmacology , COVID-19/pathology , Carboxypeptidases/drug effects , Cell Line , Endoplasmic Reticulum/metabolism , Glycosylation/drug effects , HEK293 Cells , Humans , Membrane Proteins/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Swine
7.
Nat Commun ; 12(1): 4918, 2021 08 13.
Article in English | MEDLINE | ID: covidwho-1397870

ABSTRACT

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.


Subject(s)
Cell Nucleolus/genetics , DNA Repair , DNA, Ribosomal/genetics , Saccharomyces cerevisiae Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/genetics , Valosin Containing Protein/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleolus/metabolism , DNA Damage , DNA, Ribosomal/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Two-Hybrid System Techniques , Valosin Containing Protein/metabolism
8.
Nat Commun ; 12(1): 4584, 2021 07 28.
Article in English | MEDLINE | ID: covidwho-1387354

ABSTRACT

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Antigens, Differentiation/genetics , Membrane Proteins/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/pharmacology , Antigens, Differentiation/metabolism , Binding Sites , COVID-19/virology , Gene Expression Regulation , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Interferon-beta/pharmacology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment/drug effects
9.
J Biol Chem ; 295(36): 12686-12696, 2020 09 04.
Article in English | MEDLINE | ID: covidwho-1387615

ABSTRACT

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.


Subject(s)
Epithelial Cells/metabolism , Membrane Proteins/metabolism , Proteolysis , Serine Proteases/metabolism , A549 Cells , Cells, Cultured , HEK293 Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mutation , Protein Domains , Protein Transport , Respiratory Mucosa/cytology , Serine Proteases/chemistry , Serine Proteases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Trypsin/metabolism
11.
Am J Respir Cell Mol Biol ; 65(3): 300-308, 2021 09.
Article in English | MEDLINE | ID: covidwho-1381187

ABSTRACT

Endothelial dysfunction is implicated in the thrombotic events reported in patients with coronavirus disease (COVID-19), but the underlying molecular mechanisms are unknown. Circulating levels of the coagulation cascade activator PAI-1 are substantially higher in patients with COVID-19 with severe respiratory dysfunction than in patients with bacterial sepsis and acute respiratory distress syndrome. Indeed, the elevation of PAI-1 is recognized as an early marker of endothelial dysfunction. Here, we report that the rSARS-CoV-2-S1 (recombinant severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] viral envelope spike) glycoprotein stimulated robust production of PAI-1 by human pulmonary microvascular endothelial cells (HPMECs). We examined the role of protein degradation in this SARS-CoV-2-S1 induction of PAI-1 and found that the proteasomal degradation inhibitor bortezomib inhibited SARS-CoV-2-S1-mediated changes in PAI-1. Our data further show that bortezomib upregulated KLF2, a shear-stress-regulated transcription factor that suppresses PAI-1 expression. Aging and metabolic disorders are known to increase mortality and morbidity in patients with COVID-19. We therefore examined the role of ZMPSTE24 (zinc metallopeptidase STE24), a metalloprotease with a demonstrated role in host defense against RNA viruses that is decreased in older individuals and in metabolic syndrome, in the induction of PAI-1 in HPMECs by SARS-CoV-2-S1. Indeed, overexpression of ZMPSTE24 blunted enhancement of PAI-1 production in spike protein-exposed HPMECs. In addition, we found that membrane expression of the SARS-CoV-2 entry receptor ACE2 was reduced by ZMPSTE24-mediated cleavage and shedding of the ACE2 ectodomain, leading to accumulation of ACE2 decoy fragments that may bind SARS-CoV-2. These data indicate that decreases in ZMPSTE24 with age and comorbidities may increase vulnerability to vascular endothelial injury by SARS-CoV-2 viruses and that enhanced production of endothelial PAI-1 might play role in prothrombotic events in patients with COVID-19.


Subject(s)
COVID-19/virology , Endothelial Cells/pathology , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Pulmonary Artery/pathology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Aging , COVID-19/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/virology , Humans , Membrane Proteins/genetics , Metalloendopeptidases/genetics , Plasminogen Activator Inhibitor 1/genetics , Proteolysis , Pulmonary Artery/metabolism , Pulmonary Artery/virology , Spike Glycoprotein, Coronavirus/genetics
12.
J Virol ; 95(14): e0011121, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1358015

ABSTRACT

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.


Subject(s)
Ferrets , Influenza A Virus, H1N1 Subtype , Interferon-alpha/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Orthomyxoviridae Infections/immunology , Animals , Cell Line , Conserved Sequence , Disease Models, Animal , Ferrets/immunology , Ferrets/metabolism , Ferrets/virology , Humans , Models, Molecular , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/metabolism , Polymerase Chain Reaction , Sequence Analysis, Protein , Up-Regulation
13.
Commun Biol ; 4(1): 366, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1351981

ABSTRACT

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins, depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.


Subject(s)
Chromatography, Gel , Green Fluorescent Proteins/metabolism , Membrane Proteins/metabolism , Nanotechnology , Peptides/metabolism , Single-Domain Antibodies/immunology , Animals , Cryoelectron Microscopy , Cysteine Loop Ligand-Gated Ion Channel Receptors/genetics , Cysteine Loop Ligand-Gated Ion Channel Receptors/immunology , Cysteine Loop Ligand-Gated Ion Channel Receptors/metabolism , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/immunology , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/immunology , Oryzias/genetics , Oryzias/metabolism , Peptides/genetics , Peptides/immunology , Protein Stability , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Spectrometry, Fluorescence , Temperature , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism
14.
Nat Cell Biol ; 23(8): 846-858, 2021 08.
Article in English | MEDLINE | ID: covidwho-1309445

ABSTRACT

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.


Subject(s)
Autophagy-Related Proteins/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , Autophagosomes/metabolism , Autophagy-Related Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , Immunoprecipitation , Membrane Proteins/genetics , Microscopy, Confocal , Protein Transport/physiology , Vesicular Transport Proteins/genetics
15.
Front Endocrinol (Lausanne) ; 12: 665134, 2021.
Article in English | MEDLINE | ID: covidwho-1305637

ABSTRACT

Introduction: Neuropilin-1(NRP1) is a cofactor that enhances SARS-CoV-2 coronavirus cell infectivity when co-expressed with angiotensin-converting enzyme 2(ACE2). The Renin-Angiotensin System (RAS) is activated in type 2 diabetes (T2D); therefore, the aim of this study was to determine if hypoglycaemia-induced stress in T2D would potentiate serum NRP1(sNRP1) levels, reflecting an increased risk for SARS-CoV-2 infection. Methods: A case-control study of aged-matched T2D (n = 23) and control (n = 23) subjects who underwent a hyperinsulinemic clamp over 1-hour to hypoglycemia(<40mg/dl) with subsequent timecourse of 4-hours and 24-hours. Slow Off-rate Modified Aptamer (SOMA)-scan plasma protein measurement determined RAS-related proteins: renin (REN), angiotensinogen (AGT), ACE2, soluble NRP1(sNRP1), NRP1 ligands (Vascular endothelial growth factor, VEGF and Class 3 Semaphorins, SEM3A) and NRP1 proteolytic enzyme (A Disintegrin and Metalloproteinase 9, ADAM9). Results: Baseline RAS overactivity was present with REN elevated and AGT decreased in T2D (p<0.05); ACE2 was unchanged. Baseline sNRP1, VEGF and ADAM9 did not differ between T2D and controls and remained unchanged in response to hypoglycaemia. However, 4-hours post-hypoglycemia, sNRP1, VEGF and ADAM9 were elevated in T2D(p<0.05). SEMA3A was not different at baseline; at hypoglycemia, SEMA3A decreased in controls only. Post-hypoglycemia, SEMA3A levels were higher in T2D versus controls. sNRP1 did not correlate with ACE2, REN or AGT. T2D subjects stratified according to ACE inhibitor (ACEi) therapies showed no difference in sNRP1 levels at either glucose normalization or hypoglycaemia. Conclusion: Hypoglycemia potentiated both plasma sNRP1 level elevation and its ligands VEGF and SEMA3A, likely through an ADAM9-mediated mechanism that was not associated with RAS overactivity or ACEi therapy; however, whether this is protective or promotes increased risk for SARS-CoV-2 infection in T2D is unclear. Clinical Trial Registration: https://clinicaltrials.gov, identifier NCT03102801.


Subject(s)
ADAM Proteins/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Diabetes Mellitus, Type 2/metabolism , Hypoglycemia/metabolism , Membrane Proteins/metabolism , Neuropilin-1/metabolism , Semaphorin-3A/metabolism , Vascular Endothelial Growth Factor A/metabolism , Aged , Angiotensins/metabolism , COVID-19 , Female , Glucose Clamp Technique , Humans , Male , Middle Aged , Protective Factors , Renin/metabolism , Risk Factors , SARS-CoV-2
16.
Int J Mol Sci ; 22(13)2021 Jul 04.
Article in English | MEDLINE | ID: covidwho-1304672

ABSTRACT

Cardiovascular diseases have attracted our full attention not only because they are the main cause of mortality and morbidity in many countries but also because the therapy for and cure of these maladies are among the major challenges of the medicine in the 21st century [...].


Subject(s)
Cardiovascular Diseases/etiology , Animals , Cardiovascular Diseases/genetics , Cardiovascular Diseases/metabolism , Complement C3/genetics , Complement C3/metabolism , Extracellular Vesicles/metabolism , Genetic Markers , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Models, Cardiovascular , Myosin Light Chains/genetics , Myosin Light Chains/metabolism , Risk Factors
17.
Bioengineered ; 12(1): 2836-2850, 2021 12.
Article in English | MEDLINE | ID: covidwho-1297360

ABSTRACT

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/etiology , Membrane Proteins/genetics , SARS-CoV-2 , Serine Endopeptidases/genetics , Tupaiidae/genetics , Tupaiidae/metabolism , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Animals , Bioengineering , COVID-19/enzymology , COVID-19/genetics , Computational Biology , Disease Models, Animal , Female , Humans , Male , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Molecular , Phylogeny , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Structural Homology, Protein , Tissue Distribution , Tupaiidae/virology
18.
Methods Mol Biol ; 2099: 9-20, 2020.
Article in English | MEDLINE | ID: covidwho-1292544

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic pathogen with a broad host range. The extent of MERS-CoV in nature can be traced to its adaptable cell entry steps. The virus can bind host-cell carbohydrates as well as proteinaceous receptors. Following receptor interaction, the virus can utilize diverse host proteases for cleavage activation of virus-host cell membrane fusion and subsequent genome delivery. The fusion and genome delivery steps can be completed at variable times and places, either at or near cell surfaces or deep within endosomes. Investigators focusing on the CoVs have developed several methodologies that effectively distinguish these different cell entry pathways. Here we describe these methods, highlighting virus-cell entry factors, entry inhibitors, and viral determinants that specify the cell entry routes. While the specific methods described herein were utilized to reveal MERS-CoV entry pathways, they are equally suited for other CoVs, as well as other protease-dependent viral species.


Subject(s)
Coronavirus Infections/virology , Genome, Viral/genetics , Middle East Respiratory Syndrome Coronavirus/physiology , Virus Internalization , Cell Membrane/virology , Endosomes/virology , HEK293 Cells , Humans , Membrane Proteins/metabolism , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Peptide Hydrolases/metabolism , RNA-Binding Proteins/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
19.
Basic Res Cardiol ; 116(1): 42, 2021 07 05.
Article in English | MEDLINE | ID: covidwho-1293364

ABSTRACT

Coronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.


Subject(s)
Endoplasmic Reticulum/virology , Endothelial Cells/virology , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/metabolism , Calnexin/metabolism , Cells, Cultured , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Endothelial Cells/metabolism , Host-Pathogen Interactions , Humans , Membrane Proteins/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
20.
J Virol ; 95(14): e0011121, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1287245

ABSTRACT

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.


Subject(s)
Ferrets , Influenza A Virus, H1N1 Subtype , Interferon-alpha/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Orthomyxoviridae Infections/immunology , Animals , Cell Line , Conserved Sequence , Disease Models, Animal , Ferrets/immunology , Ferrets/metabolism , Ferrets/virology , Humans , Models, Molecular , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/metabolism , Polymerase Chain Reaction , Sequence Analysis, Protein , Up-Regulation
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