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1.
Microbiol Spectr ; 10(1): e0061821, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1622002

ABSTRACT

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoprotein but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we show the antiviral spectrum of MARCH8 using viruses pseudotyped with a variety of viral envelope glycoproteins. Infection experiments revealed that viral envelope glycoproteins derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelope glycoproteins by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor. IMPORTANCE A member of the MARCH E3 ubiquitin ligase family, MARCH8, downregulates many different kinds of host transmembrane proteins, resulting in the regulation of cellular homeostasis. On the other hands, MARCH8 acts as an antiviral factor when it binds to and downregulates HIV-1 envelope glycoprotein and vesicular stomatitis virus G-glycoprotein that are viral transmembrane proteins. This study reveals that, as in the case of cellular membrane proteins, MARCH8 shows broad-spectrum inhibition against various viral envelope glycoproteins by recognizing their cytoplasmic lysine residues, resulting in lysosomal degradation.


Subject(s)
Antiviral Agents/pharmacology , Lysine/drug effects , Ubiquitin-Protein Ligases/pharmacology , Viral Envelope Proteins/chemistry , Blotting, Western , Down-Regulation , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Lysine/metabolism , Ubiquitination/physiology , Viral Envelope Proteins/drug effects
2.
Sci Rep ; 11(1): 23196, 2021 12 01.
Article in English | MEDLINE | ID: covidwho-1545648

ABSTRACT

Here, we aimed to evaluate the clinical performance of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit designed to detect the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This kit comprises automated chemiluminescence detection systems. Western blot analysis confirmed that anti-SARS-CoV antibodies detected SARS-CoV-2N proteins. The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR). The HISCL Antigen assay kit showed a sensitivity of 95.4% and 16.6% in samples with copy numbers > 100 and < 99, respectively. The kit did not cross-react with human coronaviruses causing seasonal common cold and influenza, and none of the 69 individuals without COVID-19 were diagnosed with positive results. Importantly, 81.8% of the samples with low virus load (< 50 copy numbers) were diagnosed as negative. Thus, using HISCL antigen assay kits may reduce overdiagnosis compared with RT-qPCR tests. The rapid and high-throughput HISCL SARS-CoV-2 Antigen assay kit developed here proved suitable for screening infectious COVID-19 and may help control the pandemic.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/immunology , Immunoassay/methods , SARS-CoV-2/immunology , Blotting, Western , COVID-19/immunology , COVID-19/virology , Cross Reactions , Humans , Phosphoproteins/immunology
3.
J Med Virol ; 93(12): 6671-6685, 2021 12.
Article in English | MEDLINE | ID: covidwho-1544318

ABSTRACT

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide spectrum of syndromes involving multiple organ systems and is primarily mediated by viral spike (S) glycoprotein through the receptor-binding domain (RBD) and numerous cellular proteins including ACE2, transmembrane serine protease 2 (TMPRSS2), kidney injury molecule-1 (Kim-1), and neuropilin-1 (NRP-1). In this study, we examined the entry tropism of SARS-CoV-2 and SARS-CoV using S protein-based pseudoviruses to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, digestive, reproductive, and immune systems. At least one cell line or type of primary cell from each organ system was infected by both pseudoviruses. Infection by pseudoviruses is effectively blocked by S1, RBD, and ACE2 recombinant proteins, and more weakly by Kim-1 and NRP-1 recombinant proteins. Furthermore, cells with robust SARS-CoV-2 pseudovirus infection had strong expression of either ACE2 or Kim-1 and NRP-1 proteins. ACE2 glycosylation appeared to be critical for the infections of both viruses as there was a positive correlation between infectivity of either SARS-CoV-2 or SARS-CoV pseudovirus with the level of glycosylated ACE2 (gly-ACE2). These results reveal that SARS-CoV-2 cell entry could be mediated by either an ACE2-dependent or -independent mechanism, thus providing a likely molecular basis for its broad tropism for a wide variety of cell types.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Gastrointestinal Tract/virology , Genitalia/virology , Hepatitis A Virus Cellular Receptor 1/metabolism , Immune System/virology , Neuropilin-1/metabolism , Respiratory System/virology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization , Blotting, Western , COVID-19/metabolism , COVID-19/virology , Cell Line , Cells, Cultured , Fluorescent Antibody Technique , Gastrointestinal Tract/cytology , Genitalia/cytology , Humans , Immune System/cytology , Respiratory System/cytology
4.
Int J Biol Sci ; 17(12): 3224-3238, 2021.
Article in English | MEDLINE | ID: covidwho-1524470

ABSTRACT

Mechanisms of breast cancer progression and invasion, often involve alteration of hormonal signaling, and upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Herein, we describe a rationally designed first-in-class novel small molecule inhibitor for targeting oncogenic and hormonal signaling in ER-positive breast cancer. BC-N102 treatment exhibits dose-dependent cytotoxic effects against ER+ breast cancer cell lines. BC-N102 exhibited time course- and dose-dependent cell cycle arrest via downregulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-Akt, CDK2, and CDK4 while increasing p38 mitogen-activated protein kinase (MAPK), and mineralocorticoid receptor (MR) signaling in breast cancer cell line. In addition, we found that BC-N102 suppressed breast cancer tumorigenesis in vivo and prolonged the survival of animals. Our results suggest that the proper application of BC-N102 may be a beneficial chemotherapeutic strategy for ER+ breast cancer patients.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins/metabolism , G1 Phase/drug effects , Receptors, Estrogen/metabolism , Resting Phase, Cell Cycle/drug effects , Animals , Biomarkers, Tumor/genetics , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Division , Cell Line, Tumor , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 4/genetics , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic/physiology , Humans , Maximum Tolerated Dose , Mice , Mice, Nude , Xenograft Model Antitumor Assays
6.
Curr Issues Mol Biol ; 43(3): 1212-1225, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1438531

ABSTRACT

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


Subject(s)
Antigens, Differentiation/metabolism , CD47 Antigen/metabolism , COVID-19/epidemiology , COVID-19/metabolism , Pandemics , Receptors, Immunologic/metabolism , SARS-CoV-2/metabolism , Severity of Illness Index , Signal Transduction/immunology , Blood Donors , Blotting, Western/methods , Bronchi/cytology , COVID-19/pathology , COVID-19/virology , Caco-2 Cells , Epithelial Cells/metabolism , Epithelial Cells/virology , Healthy Volunteers , Humans , Monocytes/metabolism , Monocytes/virology , Polymerase Chain Reaction/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
8.
mBio ; 12(5): e0131621, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1406604

ABSTRACT

Coronaviruses (CoVs) are emergent pathogens that may cause life-threatening respiratory diseases in humans. Understanding of CoV-host interactions may help to identify novel therapeutic targets. MOV10 is an RNA helicase involved in different steps of cellular RNA metabolism. Both MOV10 antiviral and proviral activities have been described in a limited number of viruses, but this protein has not been previously associated with CoVs. We found that during Middle East respiratory syndrome coronavirus (MERS-CoV) infection, MOV10 aggregated in cytoplasmic structures colocalizing with viral nucleocapsid (N) protein. MOV10-N interaction was confirmed by endogenous MOV10 coimmunoprecipitation, and the presence of other cellular proteins was also detected in MOV10 complexes. MOV10 silencing significantly increased both N protein accumulation and virus titer, with no changes in the accumulation of viral RNAs. Moreover, MOV10 overexpression caused a 10-fold decrease in viral titers. These data indicated that MOV10 has antiviral activity during MERS-CoV infection. We postulated that this activity could be mediated by viral RNA sequestration, and in fact, RNA immunoprecipitation data showed the presence of viral RNAs in the MOV10 cytoplasmic complexes. Expression of wild-type MOV10 or of a MOV10 mutant without helicase activity in MOV10 knockout cell lines, developed by CRISPR-Cas technology, indicated that the helicase activity of MOV10 was required for its antiviral effect. Interestingly MOV10-N interaction was conserved in other mildly or highly pathogenic human CoVs, including the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), although MOV10 antiviral activity was found only in highly pathogenic CoVs, suggesting a potential role of MOV10 in the modulation of human CoVs pathogenesis. IMPORTANCE Coronaviruses (CoVs) are emerging pathogens causing life-threatening diseases in humans. Knowledge of virus-host interactions and viral subversion mechanisms of host pathways is required for the development of effective countermeasures against CoVs. The interaction between cellular RNA helicase MOV10 and nucleocapsid (N) protein from several human CoVs is shown. Using MERS-CoV as a model, we demonstrate that MOV10 has antiviral function, requiring its helicase activity, most likely mediated by viral RNA sequestration in cytoplasmic ribonucleoprotein structures. Furthermore, we found that MOV10 antiviral activity may act only in highly pathogenic human CoVs, suggesting a role for MOV10 in modulating CoVs pathogenesis. The present study uncovers a complex network of viral and cellular RNAs and proteins interaction modulating the antiviral response against CoVs.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Nucleocapsid Proteins/metabolism , RNA Helicases/metabolism , RNA Helicases/pharmacology , Animals , Blotting, Western , Cell Line, Tumor , Chlorocebus aethiops , Fluorescent Antibody Technique , Humans , Immunoprecipitation , RNA, Viral/metabolism , Vero Cells , Virus Replication/drug effects
9.
Invest Ophthalmol Vis Sci ; 62(7): 6, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1388618

ABSTRACT

Purpose: To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. Methods: Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. Results: We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. Conclusions: Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Diabetic Retinopathy/enzymology , Receptors, Virus/metabolism , Retina/enzymology , SARS-CoV-2/physiology , Adult , Aged , Aged, 80 and over , Binding Sites , Blotting, Western , Cells, Cultured , Diabetic Nephropathies/enzymology , Diabetic Nephropathies/pathology , Diabetic Nephropathies/virology , Diabetic Retinopathy/pathology , Diabetic Retinopathy/virology , Endothelium, Vascular/enzymology , Endothelium, Vascular/virology , Female , Fluorescent Antibody Technique, Indirect , Humans , Immunohistochemistry , Male , Middle Aged , Pericytes/enzymology , Pericytes/virology , Retinal Vessels/enzymology , Retinal Vessels/pathology , Retinal Vessels/virology , Serine Endopeptidases/metabolism
10.
Respir Res ; 21(1): 154, 2020 Jun 18.
Article in English | MEDLINE | ID: covidwho-1331943

ABSTRACT

Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.


Subject(s)
Coronavirus Infections/epidemiology , Electronic Nicotine Delivery Systems , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/epidemiology , Pneumonia/metabolism , Vaping/adverse effects , alpha7 Nicotinic Acetylcholine Receptor/genetics , Angiotensin-Converting Enzyme 2 , Animals , Blood Gas Analysis , Blotting, Western , Bronchoalveolar Lavage Fluid , COVID-19 , Cytokines/analysis , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pandemics , Pneumonia/physiopathology , Random Allocation , Reference Values , Role , Severe Acute Respiratory Syndrome/epidemiology , Signal Transduction/genetics
11.
J Med Virol ; 93(12): 6671-6685, 2021 12.
Article in English | MEDLINE | ID: covidwho-1330343

ABSTRACT

Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide spectrum of syndromes involving multiple organ systems and is primarily mediated by viral spike (S) glycoprotein through the receptor-binding domain (RBD) and numerous cellular proteins including ACE2, transmembrane serine protease 2 (TMPRSS2), kidney injury molecule-1 (Kim-1), and neuropilin-1 (NRP-1). In this study, we examined the entry tropism of SARS-CoV-2 and SARS-CoV using S protein-based pseudoviruses to infect 22 cell lines and 3 types of primary cells isolated from respiratory, urinary, digestive, reproductive, and immune systems. At least one cell line or type of primary cell from each organ system was infected by both pseudoviruses. Infection by pseudoviruses is effectively blocked by S1, RBD, and ACE2 recombinant proteins, and more weakly by Kim-1 and NRP-1 recombinant proteins. Furthermore, cells with robust SARS-CoV-2 pseudovirus infection had strong expression of either ACE2 or Kim-1 and NRP-1 proteins. ACE2 glycosylation appeared to be critical for the infections of both viruses as there was a positive correlation between infectivity of either SARS-CoV-2 or SARS-CoV pseudovirus with the level of glycosylated ACE2 (gly-ACE2). These results reveal that SARS-CoV-2 cell entry could be mediated by either an ACE2-dependent or -independent mechanism, thus providing a likely molecular basis for its broad tropism for a wide variety of cell types.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Gastrointestinal Tract/virology , Genitalia/virology , Hepatitis A Virus Cellular Receptor 1/metabolism , Immune System/virology , Neuropilin-1/metabolism , Respiratory System/virology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Virus Internalization , Blotting, Western , COVID-19/metabolism , COVID-19/virology , Cell Line , Cells, Cultured , Fluorescent Antibody Technique , Gastrointestinal Tract/cytology , Genitalia/cytology , Humans , Immune System/cytology , Respiratory System/cytology
13.
Methods Mol Biol ; 2099: 21-37, 2020.
Article in English | MEDLINE | ID: covidwho-1292545

ABSTRACT

The coronavirus spike envelope glycoprotein is an essential viral component that mediates virus entry events. Biochemical assessment of the spike protein is critical for understanding structure-function relationships and the roles of the protein in the viral life cycle. Coronavirus spike proteins are typically proteolytically processed and activated by host cell enzymes such as trypsin-like proteases, cathepsins, or proprotein-convertases. Analysis of coronavirus spike proteins by western blot allows the visualization and assessment of proteolytic processing by endogenous or exogenous proteases. Here, we present a method based on western blot analysis to investigate spike protein proteolytic cleavage by transient transfection of HEK-293 T cells allowing expression of the spike protein of the highly pathogenic Middle East respiratory syndrome coronavirus in the presence or absence of a cellular trypsin-like transmembrane serine protease, matriptase. Such analysis enables the characterization of cleavage patterns produced by a host protease on a coronavirus spike glycoprotein.


Subject(s)
Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Blotting, Western , Cell Line , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Protein Processing, Post-Translational , Proteolysis , Serine Endopeptidases/metabolism , Virus Internalization
14.
Invest Ophthalmol Vis Sci ; 62(7): 25, 2021 06 01.
Article in English | MEDLINE | ID: covidwho-1280514

ABSTRACT

Purpose: The ocular surface is considered an important route for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. The expression level of the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is vital for viral infection. However, the regulation of ACE2 expression on the ocular surface is still unknown. We aimed to determine the change in ACE2 expression in inflamed corneal epithelium and explore potential drugs to reduce the expression of ACE2 on the ocular surface. Methods: The expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in human corneal epithelial cells (HCECs) was examined by qPCR and Western blotting. The altered expression of ACE2 in inflammatory corneal epithelium was evaluated in TNFα- and IL-1ß-stimulated HCECs and inflamed mouse corneal epithelium, and the effect of resveratrol on ACE2 expression in HCECs was detected by immunofluorescence and Western blot analysis. Results: ACE2 and TMPRSS2 are expressed on the human corneal epithelial cells. ACE2 expression is upregulated in HCECs by stimulation with TNFα and IL-1ß and inflamed mouse corneas, including dry eye and alkali-burned corneas. In addition, resveratrol attenuates the increased expression of ACE2 induced by TNFα in HCECs. Conclusions: This study demonstrates that ACE2 is highly expressed in HCECs and can be upregulated by stimulation with inflammatory cytokines and inflamed mouse corneal epithelium. Resveratrol may be able to reduce the increased expression of ACE2 on the inflammatory ocular surface. Our work suggests that patients with an inflammatory ocular surface may display higher ACE2 expression, which increases the risk of SARS-CoV-2 infection.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Enzyme Inhibitors/pharmacology , Epithelium, Corneal/enzymology , Gene Expression Regulation, Enzymologic/physiology , Keratitis/enzymology , Resveratrol/pharmacology , SARS-CoV-2/physiology , Adult , Angiotensin-Converting Enzyme 2/metabolism , Animals , Blotting, Western , Cells, Cultured , Epithelium, Corneal/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Humans , Inflammation/drug therapy , Inflammation/enzymology , Interleukin-1beta/pharmacology , Keratitis/drug therapy , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Fluorescence , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Virus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation
15.
Emerg Microbes Infect ; 10(1): 894-904, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1209882

ABSTRACT

Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the ongoing large-scale vaccination in different countries and continuous surge of new variants of global concerns, a convenient, cost-effective and high-throughput neutralization test is urgently needed. Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential. In this study we generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein reporter to develop neutralization assays. Similar tropism, infection kinetics and mechanism of entry through receptor-mediated endocytosis were found in the three pseudoviruses generated. Compared with pseudovirus D614, pseudovirus with D614G mutation had decreased shedding and higher density of S1 protein present on particles. The 50% neutralization titers to pseudoviruses D614 or D614G correlated with the plaque reduction neutralization titers to live SARS-CoV-2. The turn-around time of 48-72 h, minimal autofluorescence, one-step image quantification, expandable to 384-well, sequential readouts and dual quantifications by flow cytometry support its high-throughput and versatile applications at a non-reference and biosafety level 2 laboratory, in particular for assessing the neutralization sensitivity of new variants by sera from natural infection or different vaccinations during our fight against the pandemic.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , Neutralization Tests/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Ammonium Chloride/pharmacology , Animals , Antigen-Antibody Reactions , Blotting, Western , COVID-19/blood , Chlorocebus aethiops , Convalescence , Defective Viruses/genetics , Genes, Reporter , Genetic Vectors/immunology , HEK293 Cells , HIV-1/genetics , Humans , Immunoglobulin G/immunology , Lentivirus/genetics , Mutagenesis, Site-Directed , Pandemics , Point Mutation , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
16.
Food Funct ; 12(8): 3393-3404, 2021 Apr 26.
Article in English | MEDLINE | ID: covidwho-1201666

ABSTRACT

The global health emergency generated by coronavirus disease-2019 has prompted the search for immunomodulatory agents. There are many potential natural products for drug discovery and development to tackle this disease. One of these candidates is the Ganoderma lucidum fungal immunomodulatory protein (FIP-glu). In the present study, we clarify the influences of N-linked glycans on the improvement of anti-inflammatory activity and the potential mechanisms of action. Four proteins, including FIP-glu (WT) and its mutants N31S, T36N and N31S/T36N, were successfully expressed in P. pastoris, of which T36N and N31S/T36N were glycoproteins. After treatment with peptide-N-glycosidase F, the results of SDS-PAGE and Western blot showed that the glycan moiety was removed completely, indicating that the glycan moiety was N-linked. This was also demonstrated by UPLC-qTOF-MS. The cytotoxicity assay showed that N-linked glycans decreased the cytotoxicity of WT; while, the RT-qPCR assay showed that N-glycosylated WT regulated the mRNA expression of IL-6 and TGF-ß1. The Western blot results showed that N-glycosylated WT reduced the phosphorylation level of p38 MAPK. In conclusion, our findings revealed a novel mechanism by which N-glycosylation of FIP-glu improved its anti-inflammatory activity through the regulation of the expression of inflammatory cytokines in RAW264.7 via inhibition of p38 MAPK phosphorylation. It was proved that N-glycosylation significantly improved the functional properties of FIP-glu, providing theoretical and technical support for expanding the application of FIPs in the food and pharmaceutical industries.


Subject(s)
Fungal Proteins/pharmacology , Immunologic Factors/pharmacology , Immunomodulation/drug effects , Reishi , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Blotting, Western , Chromatography, High Pressure Liquid , Cytokines , Electrophoresis, Polyacrylamide Gel , Glycoproteins/metabolism , Glycosylation , Mass Spectrometry , Mice , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , RAW 264.7 Cells , Real-Time Polymerase Chain Reaction , Saccharomycetales
17.
Inflammation ; 44(5): 1865-1877, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1188133

ABSTRACT

An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNFα, IL-6, IL-1ß and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-κB p65 and IκBα, and IκBα degradation. DNA binding of NF-κB p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 µM) resulted in inhibition of spike glycoprotein S1-induced NF-κB activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment, produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNFα, IL-6, IL-1ß and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1ß production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-κB, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 are possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Cytokine Release Syndrome/virology , Cytokines/metabolism , Dexamethasone/pharmacology , Leukocytes, Mononuclear/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Anti-Inflammatory Agents/therapeutic use , Biomarkers/metabolism , Blotting, Western , COVID-19/drug therapy , COVID-19/immunology , COVID-19/virology , Cells, Cultured , Cytokine Release Syndrome/drug therapy , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/metabolism , Dexamethasone/therapeutic use , Enzyme-Linked Immunosorbent Assay , Humans , Leukocytes, Mononuclear/drug effects , Recombinant Proteins/immunology , SARS-CoV-2/immunology
18.
Eur J Clin Microbiol Infect Dis ; 40(6): 1309-1317, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1116613

ABSTRACT

ELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary serologic assay for COVID-19. The JessTM Simple Western system, an automated capillary-based assay, was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as the source of antigen, and total immunoglobulins (IgG, IgM, IgA) detection. In total, 602 sera were tested including 223 from RT-PCR-confirmed COVID-19 patients, 76 from patients diagnosed with seasonal HCoVs and 303 from coronavirus-negative control sera. We also compared this assay with the EUROIMMUN® SARS-CoV-2 IgG ELISA kit. Among 223 sera obtained from RT-PCR-confirmed COVID-19 patients, 180/223 (81%) exhibited reactivity against the nucleocapsid and 70/223 (31%) against the spike protein. Nucleocapsid reactivity was further detected in 9/76 (14%) samples collected from patients diagnosed with seasonal HCoVs and in 15/303 (5%) coronavirus-negative control samples. In the subset of sera collected more than 2 weeks after the onset of symptoms, the sensitivity was 94% and the specificity 93%, the latter value probably reflecting cross-reactivity of SARS-CoV-2 with other coronaviruses. The automated Western immunoblotting presented a substantial agreement (90%) with the compared ELISA (Cohen's Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposure of people to HCoVs including SARS-CoV-2.


Subject(s)
Antibodies, Viral/isolation & purification , Blotting, Western , COVID-19/diagnosis , Antibodies, Viral/blood , Automation, Laboratory , Coronavirus Nucleocapsid Proteins/immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Humans , Phosphoproteins/immunology , SARS-CoV-2/immunology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
19.
Nat Commun ; 12(1): 1346, 2021 03 01.
Article in English | MEDLINE | ID: covidwho-1111984

ABSTRACT

SARS-CoV-2 is the underlying cause for the COVID-19 pandemic. Like most enveloped RNA viruses, SARS-CoV-2 uses a homotrimeric surface antigen to gain entry into host cells. Here we describe S-Trimer, a native-like trimeric subunit vaccine candidate for COVID-19 based on Trimer-Tag technology. Immunization of S-Trimer with either AS03 (oil-in-water emulsion) or CpG 1018 (TLR9 agonist) plus alum adjuvants induced high-level of neutralizing antibodies and Th1-biased cellular immune responses in animal models. Moreover, rhesus macaques immunized with adjuvanted S-Trimer were protected from SARS-CoV-2 challenge compared to vehicle controls, based on clinical observations and reduction of viral loads in lungs. Trimer-Tag may be an important platform technology for scalable production and rapid development of safe and effective subunit vaccines against current and future emerging RNA viruses.


Subject(s)
COVID-19 Vaccines/therapeutic use , COVID-19/immunology , COVID-19/prevention & control , SARS-CoV-2/pathogenicity , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Blotting, Western , COVID-19/therapy , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunity, Cellular/physiology , Immunization, Passive , Immunohistochemistry , Macaca mulatta , Mice , Mice, Inbred BALB C , Microscopy, Electron , SARS-CoV-2/immunology
20.
J Clin Lab Anal ; 35(4): e23735, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1092109

ABSTRACT

BACKGROUND: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID-19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS-CoV-2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. METHODS: An enzyme-linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. RESULTS: Preliminary data from 10 sera (5 patients with COVID-19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS-CoV-2-positive individuals. CONCLUSION: This ELISA appears to be a specific and reliable method for detecting COVID-19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus.


Subject(s)
Antigens, Viral , COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2 , Virus Cultivation/methods , Animals , Antibodies, Viral/blood , Antigens, Viral/chemistry , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Blotting, Western , COVID-19/immunology , COVID-19/virology , Chlorocebus aethiops , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/immunology , Coronavirus Nucleocapsid Proteins/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Humans , Phosphoproteins/chemistry , Phosphoproteins/immunology , Phosphoproteins/isolation & purification , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/isolation & purification , Vero Cells
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