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Principal Investigator: JOHN H KEHRL
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2021
Award: $51,546
Funding agency: National Institute of Allergy and Infectious Diseases
Beta-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we have demonstrated that Beta-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of Beta-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. -Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immune abnormalities observed in patients and suggests new therapeutic modalities.
We have continued our studies of SARS CoV2 spike protein. To assess the effect of the spike protein lung vasculature homeostasis, we injected Evans blue dye into the blood and intranasally administered recombinant spike protein. At various time points the presence of Evans blue in lung tissue was assessed. The intranasal administration of the original SARS-CoV-2 spike protein rapidly (within 30 minutes) increased the mouse lung vasculature permeability, 3 fold higher than a similarly administered HKU-1 spike protein used as a control. We have also evaluated the SARS CoV2 spike protein variants E484K and N501Y. Following the intranasal inoculation into mice, lung sections have shown a pattern of cellular acquisition similar to the original spike protein. Alveolar macrophages (AMs) rapidly acquired the spike variants. The inoculation also resulted in a rapid although transient increase in lung neutrophils. In vitro binding assays with the B1.1.7 (UK) variant RBD domain or the S1 domain revealed a much higher binding affinity to human monocytes and neutrophils. By using spike protein incorporated into viral like particles (VLPs) we showed that they bound efficiently to ACE2 overexpressed HEK293 cells. In contrast, VLPs incorporated with HKU-1 and 229E spike proteins bound 50-fold less well. A comparison of the original spike protein versus UK and South African variants incorporated VLPs revealed that the variants spike VLPs bound 5 times better to ACE2 over expressed HEK293 cells than did the VLPS carrying the original spike protein. In vitro binding assay with human PBMC revealed that monocytes efficiently bound the spike protein bearing VLPs. To examine their behavior in vivo, we administered the VLPs intranasally. Thick lung section imaging revealed that alveolar macrophages were major cell population responsible for VLPs acquisition, much like previous studies with the spike protein. Surprisingly we also noted that the VLPs rapidly translocated across the alveolus epithelium into interstitial tissues, eventually entering the lymph via local lymphatic portals. The VLPs transited through the lymph too enter the primary lymph node, the TBLN (tracheobronchial lymph node). Cell population analysis of lung and TBLN by flow cytometry is in progress following nasal administration of spike protein and spike protein bearing VLPs.
The SARS-CoV-2 genome contains a unique open reading frame 8, which encodes an immunogenic secreted protein (ORF8). Extracellular ORF8 has been detected in cell culture supernatants and in the sera of COVID-19 patients. In addition, COVID-19 patients develop ORF8 reactive antibodies. The expression of ORF8 in mammalian cell lines revealed a largely cytosolic protein with some ER localization. We purified the mammalian expressed protein by affinity chromatography and gel filtration, and fluorescently labeled it. To assess ORF8 cellular interactions in vivo we intranasally, intravenously, and intradermally inoculated mice with fluorescently labeled protein. Following the intranasal injection, we noted a rapid, intense, and persistent acquisition of ORF8 by lung alveolar macrophages. Neutrophils, monocytes, lymphocytes, eosinophils, NK cells, and dendritic cells (DCs) also acquired it. Following IV injection Kupffer cells and sinusoid endothelial cells in the liver rapidly gathered the ORF8 protein in granule-like structures that lined the endothelium. Lymphatic endothelial cells and local lymph node macrophages acquired locally injected protein. Intradermal injection in the cremaster muscle of male mice caused neutrophil recruitment and led to neutrophil damage. Longitudinal analysis of mouse lungs following ORF8 protein intranasal delivery showed a rapidly expanding population of neutrophils, which gradually declined over the ensuing 24 hours, only to be replaced by monocytes and lymphocytes. Lung section imaging revealed a population of E-cadherin positive cells (potentially type II alveolar epithelial cells), which gradually acquired ORF8 protein. In vitro binding assays using human peripheral blood mononuclear cells demonstrated strong binding to human blood B cells, monocytes, and neutrophils. A population of DCs bound ORF8 while CD4, CD8, NKT, and NK cells did not. Based on the known importance of cytokines in COVID-19, we measured selected cytokines in conditioned supernatants of human M0/M1/M2 macrophages exposed to ORF8 protein. We found that ORF8 induced IL-6 and Il-10 secretion by M0 and M2 macrophages. IL-12p40, IL-12p70, CXCL10, IL-1b, IL-23, and TNF-alpha increased in the supernatants of conditioned M1 macrophages, and CCL17 and CXCL10 in M2 macrophages. CXCL10 and CCL17 production by macrophages triggered by ORF8, may explain the recruitment of leukocytes to the site of ongoing SARS-CoV2 replication. Elevated CXCL10, IL-6, and IL-10 have been reported to predict clinical progression in patients suffering from COVID-19.
Previous studies of SARS-CoV-1 identified open reading frame 3a (ORF3a) as an essential factor for disease pathogenesis. ORF3a is a transmembrane protein that contains several conserved motifs including a cysteine-rich motif. We have investigated the effect of several cysteine-to-alanine substitution mutations, the impact of the common Q57H mutation, and the effect of various C-terminal truncations. We have determined that disulfide bond formation at cysteine-133 is integral for ORF3a to oligomerize. The Q57H mutation alters the morphology of ORF3a transfected cells triggering micro-spike formation. The C-terminal truncations progressively alter ORF3a intracellular localization resulting in the confinement of the most truncated proteins to the ER. We had previously shown that SARS-CoV-1 ORF3a oligomers insert into the plasma and lysosomal membranes. Imaging studies have shown a similar localization of SARS-CoV-2 ORF3a. Several host cell proteins have been reported to interact with ORF3a. We have verified several of these results and are working to understand their functional importance. Using standard immunoprecipitation assays, imaging of fluorescently tagged proteins, and FRET-FILM studies we have shown interactions between ORF3a and the chloride channel CLCC1; the lysosomal TPC2 and TMEM16b proteins; HMOX1 (an enzyme linked to innate immunity); and the SARS-CoV-2 envelope protein. Expression of both SARS-CoV-1 and -2 ORF3a causes lysosomal damage and lysosomal deacidification, which may contribute to viral egress. SARS-CoV-2 ORF3a is also a strong inducer of HMOX1 expression. Preliminary evidence suggests ORF3a raises HMOX1 expression by interfering with its degradation. To further investigate ORF3a, we have established a collaboration with several extramural investigators, who have expertise in the study of ion channels, drug discovery, structural studies, and virology.
Terms: <(TNF)-α><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><A-152E5.3><ABCD-2><ABCD-2 protein><ACE2><Affinity><Affinity Chromatography><Alanine><Alveolar Macrophages><Alveolus><Antibodies><Antigen Presentation Pathway><Antigen Processing and Presentation><Assay><Autoregulation><Azovan Blue><B blood cells><B cell><B cell differentiation factor><B cell stimulating factor 2><B cells><B-Cell Differentiation Factor><B-Cell Differentiation Factor-2><B-Cell Stimulatory Factor-2><B-Cells><B-Lymphocytes><B-cell><B.1.1.7><B.1.351><BCDF><BSF-2><BSF2><Behavior><Binding><Binding Proteins><Bioassay><Biologic Assays><Biological Assay><Blood><Blood Cells><Blood Eosinophil><Blood Neutrophil><Blood Plasma><Blood Polymorphonuclear Neutrophil><Blood Reticuloendothelial System><Blood leukocyte><Blood monocyte><Body Tissues><Bronchial Alveolus><C-terminal><CC Chemokine TARC><CCL17><CCL17 gene><CD40 Receptor-Associated Factor 1><CD40bp Protein><CD8><CD8B><CD8B1><CD8B1 gene><COVID infected patient><COVID patient><COVID positive patient><COVID-19><COVID-19 S protein><COVID-19 genome><COVID-19 infected patient><COVID-19 pathogenesis><COVID-19 patient><COVID-19 positive patient><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID-19 virus genome><COVID19><COVID19 S protein><COVID19 genome><COVID19 pathogenesis><COVID19 patient><COVID19 positive patient><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 virus><COVID19 virus genome><CRAF1 Protein><CRG-2><CSIF><CSIF-10><CV-19><CV19><CXCL10><CXCL10 gene><Cachectin><Cadherin-1><Carbohydrates><Cell Body><Cell Communication and Signaling><Cell Culture Techniques><Cell Function><Cell Line><Cell Process><Cell Signaling><Cell Surface Receptors><Cell Survival><Cell Viability><Cell physiology><Cell-Mediated Cytolysis><Cell-Mediated Lympholysis><CellLine><Cells><Cellular Cytotoxicity><Cellular Function><Cellular Physiology><Cellular Process><Chemokine (C-C Motif) Ligand 17><Chloride Channels><Chloride Ion Channels><CoV-2><CoV2><Collaborations><Coloring Agents><Confocal Microscopy><Coronaviridae><Coronavirus><Cysteine><Cytokine Synthesis Inhibitory Factor><Cytoplasmic Granules><Cytotoxic cell><Dendritic Cells><Discontinuous Capillary><Disease><Disorder><Dyes><E-Cadherin><EXTMR><Endothelial Cells><Endothelium><Envelope Protein><Enzyme Gene><Enzymes><Eosinophilic Granulocyte><Eosinophilic Leukocyte><Epithelial><Epithelial Calcium-Dependent Adhesion Protein><Epithelial Cells><Epithelial-Cadherin><Evans Blue><Evans blue stain><Exclusion Chromatography><Exposure to><Extramural><Extramural Activities><FRET><Family><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Fluorescence Resonance Energy Transfer><Förster Resonance Energy Transfer><GTP Phosphohydrolases><GTPases><Gel Chromatography><Gel Filtration><Gel Filtration Chromatography><Gel Permeation Chromatography><Genetic Alteration><Genetic Change><Genetic defect><Guanosine Triphosphate Phosphohydrolases><Guanosinetriphosphatases><HPGF><Half-Cystine><Hepatocyte-Stimulating Factor><Homeostasis><Hour><Human><Human Cell Line><Hybridoma Growth Factor><IFI10><IFN-beta 2><IFNB2><IL-10><IL-23><IL-6><IL10><IL10A><IL6 Protein><INP10><IP-10><Image><Immune><Immune Precipitation><Immune response><Immunes><Immunological response><Immunoprecipitation><In Vitro><Inflammasome><Inflammation><Inflammatory><Injections><Innate Immunity><Integral Membrane Protein><Interleukin 10 Precursor><Interleukin-10><Interleukin-6><Intracellular Communication and Signaling><Intranasal Administration><Intranasal Drug Administration><Intravenous><Intrinsic Membrane Protein><Investigators><Ion Channel><Ionic Channels><K lymphocyte><Kupffer Cells><L-Cysteine><LAP-1 Protein><LYT3><Label><Leukocytes><Leukocytes Reticuloendothelial System><Ligand Binding Protein><Ligand Binding Protein Gene><Link><Liver><Lung><Lung Alveolar Epithelia><Lung Parenchyma><Lung Respiratory System><Lung Tissue><Lymph><Lymph Node Reticuloendothelial System><Lymph node proper><Lymphatic><Lymphatic Endothelial Cells><Lymphatic cell><Lymphatic nodes><Lymphocyte><Lymphocyte Cytotoxicity><Lymphocytic><Lymphocytotoxicity><Lysosomes><MGI-2><MOB-1><Macrophage-Derived TNF><Mammalian Cell><Marrow Eosinophil><Marrow Neutrophil><Marrow leukocyte><Marrow monocyte><Measures><Mediating><Membrane><Membrane Channels><Metabolic Glycosylation><Methodology><Mice><Mice Mammals><Mitochondria><Modality><Modern Man><Molecular><Molecular Interaction><Monocyte-Derived TNF><Monomeric G-Proteins><Monomeric GTP-Binding Proteins><Morphology><Murine><Mus><Mutation><Myeloid Differentiation-Inducing Protein><Mφ><NK Cells><Nasal><Nasal Passages Nose><Native Immunity><Natural Immunity><Natural Killer Cells><Neutrophil Infiltration><Neutrophil Recruitment><Neutrophilic Granulocyte><Neutrophilic Leukocyte><Non-Specific Immunity><Non-lytic><Non-structural Protein><Nonlytic><Nonspecific Immunity><Nonstructural Protein><Nose><ORFs><Open Reading Frames><Organelles><PBMC><Pathogenesis><Pathogenicity><Pathway interactions><Patients><Pattern><Peripheral Blood Cell><Peripheral Blood Mononuclear Cell><Permeability><Physiological Homeostasis><Plasma><Plasma Serum><Plasmacytoma Growth Factor><Polymorphonuclear Cell><Polymorphonuclear Leukocytes><Polymorphonuclear Neutrophils><Population><Population Analysis><Preparation><Production><Property><Protein Binding><Protein Coding Region><Proteins><Proteomics><Pulmonary Macrophages><RNA Seq><RNA Viruses><RNA sequencing><RNAseq><Receptor Protein><Recombinant Proteins><Recombinants><Reporter><Reporting><Research Personnel><Researchers><Respiratory System, Nose, Nasal Passages><Reticuloendothelial System, Serum, Plasma><Role><Route><SARS><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS coronavirus disease><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV disease><SARS-CoV-2><SARS-CoV-2 B.1.1.7><SARS-CoV-2 B.1.351><SARS-CoV-2 S protein><SARS-CoV-2 genome><SARS-CoV-2 infected patient><SARS-CoV-2 patient><SARS-CoV-2 positive patient><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 genome><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SCYA17><SCYB10><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome coronavirus disease><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Sinusoid><Sinusoidal Capillary><Site><Small G-Proteins><Small GTPases><Small Inducible Cytokine A17><Small Inducible Cytokine Subfamily A (Cys-Cys), Member 17><South Africa variant><South African variant><Spleen><Spleen Reticuloendothelial System><Stellate Sinusoidal Macrophage><Strains Cell Lines><Structure><Structure of parenchyma of lung><Subcellular Process><TARC><TNF><TNF A><TNF Alpha><TNF Receptor-Associated Factor 6 Gene><TNF gene><TNF receptor-associated factor 3><TNF-α><TNFA><TNFα><TRAF-3><TRAF3><TRAF6><TRAF6 gene><Techniques><Thick><Thickness><Time><Tissues><Tracheobronchial><Transgenic Mice><Transmembrane Protein><Transmembrane Protein Gene><Tumor Necrosis Factor><Tumor Necrosis Factor-alpha><U.K. variant><UK variant><United Kingdom variant><Uvomorulin><Vaccine Design><Variant><Variation><Veiled Cells><Viral><Virus><Virus Replication><White Blood Cells><White Cell><Wuhan coronavirus><affinity purification><alveolar epithelium><angiotensin converting enzyme 2><angiotensin converting enzyme II><base><beta CoV><beta coronavirus><betaCoV><betacoronavirus><biological signal transduction><bound protein><cell culture><cell mediated cytotoxicity><cell type><clinical predictors><corona virus><corona virus disease 2019><coronavirus disease 2019><coronavirus disease 2019 S protein><coronavirus disease 2019 genome><coronavirus disease 2019 infected patient><coronavirus disease 2019 pathogenesis><coronavirus disease 2019 patient><coronavirus disease 2019 positive patient><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 virus><coronavirus disease 2019 virus genome><coronavirus disease infected patient><coronavirus disease patient><coronavirus disease positive patient><coronavirus patient><cremaster muscle><cultured cell line><cytokine><disulfide bond><drug discovery><env Antigens><env Gene Products><env Polyproteins><env Protein><eosinophil><exposed human population><extracellular><flow cytophotometry><fluorescence imaging><fluorescent imaging><gIP-10><genome mutation><glycosylation><granule><guanosinetriphosphatase><hCoV19><hepatic body system><hepatic organ system><host response><human exposure><imaging><imaging study><immune system response><immunogenic><immunoresponse><in vitro Model><in vivo><inhibitor><inhibitor/antagonist><insight><interferon beta 2><interleukin-23><interstitial><intra-vital microscopy><intradermal injection><intravital microscopy><liver macrophage><longitudinal analysis><lymph cell><lymph gland><lymph nodes><lymphatic fluid><lymphnodes><macrophage><male><membrane structure><mitochondrial><monocyte><nCoV2><neutrophil><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel therapeutics><novel therapy><overexpress><overexpression><particle><pathway><patient infected with COVID><patient infected with COVID-19><patient infected with SARS-CoV-2><patient infected with coronavirus disease><patient infected with coronavirus disease 2019><patient infected with severe acute respiratory syndrome coronavirus 2><patient with COVID><patient with COVID-19><patient with COVID19><patient with SARS-CoV-2><patient with coronavirus disease><patient with coronavirus disease 2019><patient with severe acute respiratory distress syndrome coronavirus 2><peripheral blood><pulmonary><receptor><recruit><severe acute respiratory syndrome coronavirus 2 B.1.1.7><severe acute respiratory syndrome coronavirus 2 B.1.351><severe acute respiratory syndrome coronavirus 2 genome><severe acute respiratory syndrome coronavirus 2 infected patient><severe acute respiratory syndrome coronavirus 2 patient><severe acute respiratory syndrome coronavirus 2 positive patient><severe acute respiratory syndrome-CoV><social role><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><trafficking><transcriptome sequencing><viral multiplication><viral replication><virology><virus envelope><virus multiplication><white blood cell><white blood corpuscle><β CoV><β coronavirus><βCoV>