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Principal Investigator: JOHN H KEHRL
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2022
Award: $40,147
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). Our recent studies have focused on two SARS-CoV-2 encoded proteins, open reading frame (ORF) 3a and 8. Previous studies of SARS-CoV-1 identified ORF3a as an essential factor for disease pathogenesis and more recent studies with SARS-CoV-2 have reached the same conclusion. ORF3a is a transmembrane protein that contains several conserved motifs including a cysteine-rich motif. We have shown that SARS-CoV-2 ORF3a oligomers insert into the plasma and lysosomal membranes as well as some undefined cellular vesicles. Its expression in cells causes cell death via apoptosis and necrosis; lysosomal damage; increased intracellular vesicle formation; and disruption of host cell autophagy. It is also implicated in the egress of SARS-CoV-2 virions from infected cells. 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. ORF3a interacts with multiple viral and host proteins. Interactions between with ORF3a and the envelope protein (E) as well as NSP3 have been verified by co-immunoprecipitations and fluorescent lifetime imaging (FILM) microscopy. We have documented interactions between ORF3a and the following host proteins: lysosomal channel protein TCP2, the lysosomal membrane protein TMEM106, the endoplasmic reticulum (ER) and plasma membrane chloride channel CLCC1, and the ER protein HMOX1. HMOX1 is an enzyme that modulates host immune cell activity and, which can suppress viral replication and inflammatory pathways. SARS-CoV-2 ORF3a and HMOX1 co-localize in the ER. ORF3a expression stabilizes HMOX1 protein levels within cells. These results suggest that ORF3a may affect ERC8, an important E3 ligase that help direct protein traffic through the ER, and is known to ubiquitinate HMOX1, affecting its expression. TRC8 localizes on ER membrane with ORF3a. TRC8 overexpression reduces ORF3a protein levels via ER-phagy, a process related to autophagy. TRC8 also reduced other SARS-CoV2 proteins including the spike protein (S), the E protein, the matric protein (M), ORF8, and NSP3. In contrast, TRC8 had no effect on the levels of the ER localized NSP4 and NSP6, two other SARS-CoV-2 encoded proteins. Besides increasing intracellular vesicles ORF3a also enhanced extracellular vesicle production. Extracellular vesicles containing S, E, N, ORF3a, ORF7a, and ORF8 protein have been found following their expression in cell lines. ORF3a upregulated the levels of Sterol regulatory-element binding proteins (SREBPs), transcription factors that regulate genes involved in lipid synthesis providing an explanation for the increase in intracellular and extra cellular vesicles in ORF3a expressing cells. In collaborative studies recombinant E protein and ORF3a have been produced and introduced into phosopholipid bilayer nanodiscs for eventual structural studies. ORF3a and E protein have been expressed alone or together in Xenopus oocytes to assess individual channel activity and potential interactions. Finally, antibodies to ORF3a have been produced in rabbits.
The SARS-CoV-2 genome 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. When injected in mice ORF8 binds to lymphatic endothelial cells and triggers local inflammation. Since ORF8 lacks intrinsic chemoattractant activity, its injection likely triggers local chemoattractant production. Monocytes and B cells are the predominant cell populations in human PBMC that bind ORF8. ORF8 induced human bone marrow derived macrophages to secrete TNF, IL6, Il-10, IL-12, IL-1, IL-23, CXCL10, CCL17, and CXCL10. Mouse marginal zone B cells avidly bind ORF8 protein. Other B cell subsets also bind, while CD4 and CD8 T cells do not. ORF8 protein inhibits peripheral blood human B cells differentiation. The addition of ORF8 to B cell cultures did not affect cell death or early BCR signaling. It slightly reduced B cell proliferation, but it significantly reduced the generation of plasmablasts (45%), and it reduced the levels of IgG1, IgG2, IgG3, IgA, and IgM secretion. A manuscript describing this work is in progress. Consistent with an effect on B cell antibody production ORF8 protein altered important B transcription factor levels downregulating the IRF4/Pax-5 ratio as assessed by intracellular flow cytometry.
Terms: <(TNF)-α><19S Gamma Globulin><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><Ab response><Affect><Affinity Chromatography><Alanine><Antibodies><Antibody Formation><Antibody Production><Apoptosis><Apoptosis Pathway><Assay><Autophagocytosis><B Cell Proliferation><B blood cells><B cell><B cell differentiation><B cells><B lymphocyte differentiation><B-Cell Differentiation Factor Gene><B-Cell Stimulatory Factor 2 Gene><B-Cell Subsets><B-Cells><B-Lymphocyte Subsets><B-Lymphocytes><B-cell><BSF-2 Gene><BSF2 Gene><Basal Transcription Factor><Basal transcription factor genes><Beta-2 Gene Interferon><Binding><Binding Proteins><Bioassay><Biologic Assays><Biological Assay><Blood><Blood Cells><Blood Plasma><Blood Reticuloendothelial System><Blood leukocyte><Blood monocyte><Bone Marrow><Bone Marrow Reticuloendothelial System><C-terminal><CC Chemokine TARC><CCL17><CCL17 gene><CD40 Receptor-Associated Factor 1><CD40bp Protein><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><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><CV-19><CV19><CXCL10><CXCL10 gene><Cachectin><Carbohydrates><Cell Body><Cell Communication and Signaling><Cell Culture Techniques><Cell Death><Cell Function><Cell Line><Cell Process><Cell Signaling><Cell Surface Receptors><Cell Survival><Cell Viability><Cell membrane><Cell physiology><Cell-Mediated Cytolysis><Cell-Mediated Lympholysis><CellLine><Cells><Cellular Cytotoxicity><Cellular Function><Cellular Physiology><Cellular Process><Chemoattractants><Chemokine (C-C Motif) Ligand 17><Chemotactic Factors><Chemotaxins><Chloride Channels><Chloride Ion Channels><Co-Immunoprecipitations><CoV-2><CoV2><Confocal Microscopy><Cysteine><Cytoplasmic Membrane><Disease><Disorder><Domestic Rabbit><E protein><E3 Ligase><E3 Ubiquitin Ligase><Edodekin Alfa><Endoplasmic Reticulum><Endothelial Cells><Envelope Protein><Enzyme Gene><Enzymes><Ergastoplasm><Exclusion Chromatography><Family><Flow Cytofluorometries><Flow Cytofluorometry><Flow Cytometry><Flow Microfluorimetry><Flow Microfluorometry><Gel Chromatography><Gel Filtration><Gel Filtration Chromatography><Gel Permeation Chromatography><General Transcription Factor Gene><General Transcription Factors><Generations><Genes><Genetic Alteration><Genetic Change><Genetic defect><HRCA1><HSF Gene><Half-Cystine><Hepatocyte Stimulatory Factor Gene><Human><Human Cell Line><Hybridoma Growth Factor Gene><IFI10><IFNB2 Gene><IL-1><IL-12><IL-23><IL-6 Gene><IL1><IL12><IL6><IL6 gene><INP10><IP-10><IRF4><IRF4 gene><IgA><IgG1><IgG2><IgG3><IgM><Image><Immune><Immune response><Immunes><Immunoglobulin A><Immunoglobulin M><Immunological response><Individual><Inflammasome><Inflammation><Inflammatory><Injections><Innate Immunity><Integral Membrane Protein><Interferon Regulatory Factor 4><Interleukin 6 (Interferon, Beta 2) Gene><Interleukin I><Interleukin-1><Interleukin-12><Interleukin-6 Gene><Intracellular Communication and Signaling><Intravenous><Intrinsic Membrane Protein><L-Cysteine><LAP-1 Protein><LSIRF Protein><Label><Leukocytes><Leukocytes Reticuloendothelial System><Ligand Binding Protein><Ligand Binding Protein Gene><Lipids><Liver><Lung><Lung Respiratory System><Lymph Node Reticuloendothelial System><Lymph node proper><Lymphatic Endothelial Cells><Lymphatic nodes><Lymphocyte Cytotoxicity><Lymphocyte-Stimulating Hormone><Lymphocytotoxicity><MATRICS><MATRICS Consensus Cognitive Battery><MOB-1><Macrophage Cell Factor><Macrophage-Derived TNF><Mammalian Cell><Manuscripts><Marrow leukocyte><Marrow monocyte><Measurement and Treatment Research to Improve Cognition in Schizophrenia><Mediating><Membrane><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Metabolic Glycosylation><Mice><Mice Mammals><Microscopy><Mitochondria><Modern Man><Molecular><Molecular Interaction><Monocyte-Derived TNF><Morphology><Multiple Myeloma Oncogene 1><Murine><Mus><Mutation><Mφ><NKSF><NSP3><Native Immunity><Natural Immunity><Natural Killer Cell Stimulatory Factor><Necrosis><Necrotic><Non-Specific Immunity><Non-structural Protein><Nonspecific Immunity><Nonstructural Protein><ORFs><Open Reading Frames><Oryctolagus cuniculus><PBMC><Pathogenesis><Pathogenicity><Pathway interactions><Pattern><Peripheral Blood Cell><Peripheral Blood Mononuclear Cell><Plasma><Plasma Membrane><Plasma Serum><Plasmablast><Population><Preparation><Process><Production><Programmed Cell Death><Property><Protein Binding><Protein Coding Region><Proteins><Proteomics><RNA Seq><RNA Viruses><RNA sequencing><RNAseq><RNF139><RNF139 gene><Rabbits><Rabbits Mammals><Receptor Protein><Recombinant Proteins><Recombinants><Regulatory Element><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-CO-V2><SARS-COVID-2><SARS-CoV><SARS-CoV disease><SARS-CoV-1><SARS-CoV-2><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><SH2D3C><SH2D3C gene><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Coronavirus 2><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><Small Inducible Cytokine A17><Small Inducible Cytokine Subfamily A (Cys-Cys), Member 17><Spleen><Spleen Reticuloendothelial System><Sterols><Strains Cell Lines><Subcellular Process><Surface Proteins><T Helper Factor><T8 Cells><T8 Lymphocytes><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><TRC8><Techniques><Transcription Factor Proto-Oncogene><Transcription factor genes><Transgenic Mice><Transmembrane Protein><Transmembrane Protein Gene><Tumor Necrosis Factor><Tumor Necrosis Factor-alpha><Ubiquitin Protein Ligase><Ubiquitin-Protein Ligase Complexes><Ubiquitin-Protein Ligase E3><Vaccine Design><Vesicle><Viral><Virion><Virus><Virus Particle><Virus Replication><White Blood Cells><White Cell><Work><Wuhan coronavirus><Xenopus oocyte><affinity purification><angiotensin converting enzyme 2><angiotensin converting enzyme II><antibody biosynthesis><autophagy><base><beta CoV><beta coronavirus><betaCoV><betacoronavirus><biological signal transduction><bound protein><cell culture><cell cultures><cell mediated cytotoxicity><cell type><complement chemotactic factor><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 disease-19><coronavirus disease-19 pathogenesis><coronavirus disease-19 patient><coronavirus disease-19 virus><coronavirus infectious disease-19><coronavirus patient><cultured cell line><cytokine><disulfide bond><env Antigens><env Gene Products><env Polyproteins><env Protein><exposed human population><extracellular><extracellular vesicles><flow cytophotometry><gIP-10><genome mutation><glycosylation><hCoV19><hepatic body system><hepatic organ system><host response><human exposure><imaging><immune system response><immunogenic><immunoglobulin biosynthesis><immunoresponse><in vitro Model><in vivo><insight><interleukin-23><intra-vital microscopy><intravital microscopy><lymph gland><lymph nodes><lymphnodes><lymphocyte activating factor><lysosomal proteins><macrophage><membrane structure><mitochondrial><monocyte><nCoV2><nano disk><nanodisk><necrocytosis><novel><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><plasmalemma><pulmonary><receptor><recruit><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><transcription factor><transcriptome sequencing><ubiquitin-protein ligase><viral multiplication><viral replication><virus multiplication><white blood cell><white blood corpuscle><β CoV><β coronavirus><βCoV>