RESUMO
SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.
Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteólise , Replicação Viral , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
AF10, a DOT1L cofactor, is required for H3K79 methylation and cooperates with DOT1L in leukemogenesis. However, the molecular mechanism by which AF10 regulates DOT1L-mediated H3K79 methylation is not clear. Here we report that AF10 contains a "reader" domain that couples unmodified H3K27 recognition to H3K79 methylation. An AF10 region consisting of a PHD finger-Zn knuckle-PHD finger (PZP) folds into a single module that recognizes amino acids 22-27 of H3, and this interaction is abrogated by H3K27 modification. Structural studies reveal that H3 binding triggers rearrangement of the PZP module to form an H3(22-27)-accommodating channel and that the unmodified H3K27 side chain is encased in a compact hydrogen-bond acceptor-lined cage. In cells, PZP recognition of H3 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells. Together, our results uncover a pivotal role for H3K27-via readout by the AF10 PZP domain-in regulating the cancer-associated enzyme DOT1L.
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Carcinogênese/metabolismo , Regulação Leucêmica da Expressão Gênica , Histonas/metabolismo , Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , Histona-Lisina N-Metiltransferase , Histonas/química , Histonas/genética , Humanos , Ligação de Hidrogênio , Leucócitos/metabolismo , Leucócitos/patologia , Lisina/metabolismo , Metilação , Metiltransferases/química , Metiltransferases/genética , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Cells use specific mechanisms such as histone chaperones to abrogate the inherent barrier that the nucleosome poses to transcribing polymerases. The current model postulates that nucleosomes can be transiently disrupted to accommodate passage of RNA polymerases and that histones H3 and H4 possess their own chaperones dedicated to the recovery of nucleosomes. Here, we determined the crystal structure of the conserved C terminus of human Suppressors of Ty insertions 2 (hSpt2C) chaperone bound to an H3/H4 tetramer. The structural studies demonstrate that hSpt2C is bound to the periphery of the H3/H4 tetramer, mimicking the trajectory of nucleosomal-bound DNA. These structural studies have been complemented with in vitro binding and in vivo functional studies on mutants that disrupt key intermolecular contacts involving two acidic patches and hydrophobic residues on Spt2C. We show that contacts between both human and yeast Spt2C with the H3/H4 tetramer are required for the suppression of H3/H4 exchange as measured by H3K56ac and new H3 deposition. These interactions are also crucial for the inhibition of spurious transcription from within coding regions. Together, our data indicate that Spt2 interacts with the periphery of the H3/H4 tetramer and promotes its recycling in the wake of RNA polymerase.
Assuntos
Chaperonas de Histonas/metabolismo , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Complexos Multiproteicos , Nucleossomos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sais/química , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 ångström (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.
Assuntos
Endonucleases/química , Endonucleases/metabolismo , Virus da Influenza A Subtipo H5N1/enzimologia , Influenza Aviária/virologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Aves/virologia , Domínio Catalítico , Cristalografia por Raios X , Endonucleases/genética , Modelos Moleculares , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genéticaRESUMO
Immunosuppressive myeloid cells hinder immunotherapeutic efficacy in tumors, but the precise mechanisms remain undefined. Here, by performing single-cell RNA sequencing in colorectal cancer tissues, we found tumor-associated macrophages and granulocytic myeloid-derived suppressor cells increased most compared to their counterparts in normal tissue and displayed the highest immune-inhibitory signatures among all immunocytes. These cells exhibited significantly increased expression of immunoreceptor tyrosine-based inhibitory motif-bearing receptors, including SIRPA. Notably, Sirpa-/- mice were more resistant to tumor progression than wild-type mice. Moreover, Sirpα deficiency reprogramed the tumor microenvironment through expansion of TAM_Ccl8hi and gMDSC_H2-Q10hi subsets showing strong antitumor activity. Sirpa-/- macrophages presented strong phagocytosis and antigen presentation to enhance T cell activation and proliferation. Furthermore, Sirpa-/- macrophages facilitated T cell recruitment via Syk/Btk-dependent Ccl8 secretion. Therefore, Sirpα deficiency enhances innate and adaptive immune activation independent of expression of CD47 and Sirpα blockade could be a promising strategy to improve cancer immunotherapy efficacy.
Assuntos
Antígeno CD47 , Neoplasias Colorretais , Camundongos , Animais , Antígeno CD47/genética , Antígeno CD47/metabolismo , Fagocitose , Macrófagos/metabolismo , Células Mieloides/metabolismo , Neoplasias Colorretais/patologia , Microambiente TumoralRESUMO
Point mutations in the DEAD-box helicase DDX24 are associated with vascular malformations such as multi-organ venous and lymphatic defect (MOVLD) syndrome and Budd-Chiari syndrome, with the pathogenesis largely uncharacterized. DDX24 is mainly located in the nucleolus, where nucleophosmin (NPM1) regulates nucleolar homeostasis via liquid-liquid phase separation (LLPS). However, the connection between DDX24 and NPM1 in vascular malformation remains elusive. Here we demonstrated that DDX24 formed biomolecular condensates in vitro and the mutated DDX24 protein, DDX24E271K, partitioned less into the nucleoli in tissues from patients with MOVLD syndrome and cultured endothelial cells (ECs), altering nucleolar morphology. Furthermore, DDX24 was directly associated with NPM1 to regulate its phase behavior as a client in the nucleolar granular component (GC). Functionally, we showed that DDX24 was essential in maintaining nucleolar homeostasis of ECs and that either mutation or knockdown of DDX24 led to the dysfunction of ribosome biogenesis and the elevated capability of cell migration and tube formation. Our findings illustrate how DDX24 mutation affects nucleolar structure and function by regulating the phase behavior of NPM1 in the setting of vascular malformation.
Assuntos
Células Endoteliais , Malformações Vasculares , Humanos , Proteínas Mutadas de Ataxia Telangiectasia , RNA Helicases DEAD-box/genética , Homeostase/genética , Mutação/genética , NucleofosminaRESUMO
Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within individual bats, and hence the frequency of virus co-infection and spillover among them. We characterize the mammal-associated viruses in 149 individual bats sampled from Yunnan province, China, using an unbiased meta-transcriptomics approach. This reveals a high frequency of virus co-infection (simultaneous infection of bat individuals by multiple viral species) and spillover among the animals studied, which may in turn facilitate virus recombination and reassortment. Of note, we identify five viral species that are likely to be pathogenic to humans or livestock, based on phylogenetic relatedness to known pathogens or in vitro receptor binding assays. This includes a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV and SARS-CoV-2. In vitro assays indicate that this recombinant virus can utilize the human ACE2 receptor such that it is likely to be of increased emergence risk. Our study highlights the common occurrence of co-infection and spillover of bat viruses and their implications for virus emergence.
Assuntos
COVID-19 , Quirópteros , Coinfecção , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Animais , Humanos , Filogenia , SARS-CoV-2 , Viroma , China/epidemiologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genéticaRESUMO
Red fluorescent proteins are useful as morphological markers in neurons, often complementing green fluorescent protein-based probes of neuronal activity. However, commonly used red fluorescent proteins show aggregation and toxicity in neurons or are dim. We report the engineering of a bright red fluorescent protein, Crimson, that enables long-term morphological labeling of neurons without aggregation or toxicity. Crimson is similar to mCherry and mKate2 in fluorescence spectra but is 100 and 28% greater in molecular brightness, respectively. We used a membrane-localized Crimson-CAAX to label thin neurites, dendritic spines and filopodia, enhancing detection of these small structures compared to cytosolic markers.
RESUMO
BACKGROUND: Airway inflammation is the core pathological process of asthma, with the key inflammatory regulators incompletely defined. Recently, fibroblast growth factor 2 (FGF2) has been reported to be an inflammatory regulator; however, its role in asthma remains elusive. This study aimed to investigate the immunomodulatory role of FGF2 in asthma. METHODS: First, FGF2 expression was characterised in clinical asthma samples and the house dust mite (HDM)-induced mouse chronic asthma model. Second, recombinant mouse FGF2 (rm-FGF2) protein was intranasally delivered to determine the effect of FGF2 on airway inflammatory cell infiltration. Third, human airway epithelium-derived A549 cells were stimulated with either HDM or recombinant human interleukin-1ß (IL-1ß) protein combined with or without recombinant human FGF2. IL-1ß-induced IL-6 or IL-8 release levels were determined using enzyme-linked immunosorbent assay, and the involved signalling transduction was explored via Western blotting. RESULTS: Compared with the control groups, the FGF2 protein levels were significantly upregulated in the bronchial epithelium and alveolar areas of clinical asthma samples (6.70 ± 1.79 vs. 16.32 ± 2.40, P = 0.0184; 11.20 ± 2.11 vs. 21.00 ± 3.00, P = 0.033, respectively) and HDM-induced asthmatic mouse lung lysates (1.00 ± 0.15 vs. 5.14 ± 0.42, P < 0.001). Moreover, FGF2 protein abundance was positively correlated with serum total and anti-HDM IgE levels in the HDM-induced chronic asthma model (R2 = 0.857 and 0.783, P = 0.0008 and 0.0043, respectively). Elevated FGF2 protein was mainly expressed in asthmatic bronchial epithelium and alveolar areas and partly co-localised with infiltrated inflammatory cell populations in HDM-induced asthmatic mice. More importantly, intranasal instillation of rm-FGF2 aggravated airway inflammatory cell infiltration (2.45 ± 0.09 vs. 2.88 ± 0.14, P = 0.0288) and recruited more subepithelial neutrophils after HDM challenge [(110.20 ± 29.43) cells/mm2 vs. (238.10 ± 42.77) cells/mm2, P = 0.0392] without affecting serum IgE levels and Th2 cytokine transcription. In A549 cells, FGF2 was upregulated through HDM stimulation and promoted IL-1ß-induced IL-6 or IL-8 release levels (up to 1.41 ± 0.12- or 1.44 ± 0.14-fold change vs. IL-1ß alone groups, P = 0.001 or 0.0344, respectively). The pro-inflammatory effect of FGF2 is likely mediated through the fibroblast growth factor receptor (FGFR)/mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) pathway. CONCLUSION: Our findings suggest that FGF2 is a potential inflammatory modulator in asthma, which can be induced by HDM and acts through the FGFR/MAPK/NF-κB pathway in the airway epithelial cells.
Assuntos
Asma , NF-kappa B , Animais , Asma/metabolismo , Asma/patologia , Células Epiteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Inflamação/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
Fluorescent probes are vital to cell imaging by allowing specific parts of cells to be visualized and quantified. Color-switchable probes (CSPs), with tunable emission wavelength upon contact with specific targets, are particularly powerful because they not only eliminate the need to wash away all unbound probe but also allow for internal controls of probe concentrations, thereby facilitating quantification. Several such CSPs exist and have proven very useful, but not for all key cellular targets. Here we report a pioneering CSP for in situ cell imaging using aldehyde-functionalized silicon nanocrystals (SiNCs) that switch their intrinsic photoluminescence from red to blue quickly when interacting with amino acids in live cells. Though conventional probes often work better in cell-free extracts than in live cells, the SiNCs display the opposite behavior and function well and fast in universal cell lines at 37 °C while requiring much higher temperature in extracts. Furthermore, the SiNCs only disperse in cytoplasm not nucleus, and their fluorescence intensity correlated linearly with the concentration of fed amino acids. We believe these nanosilicon probes will be promising tools to visualize distribution of amino acids and potentially quantify amino acid related processes in live cells.
Assuntos
Corantes Fluorescentes , Nanopartículas , Aldeídos , Aminoácidos , Corantes Fluorescentes/química , Nanopartículas/química , SilícioRESUMO
Excessive inflammatory responses contribute to the pathogenesis and lethality of highly pathogenic human coronaviruses, but the underlying mechanism remains unclear. In this study, the N proteins of highly pathogenic human coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were found to bind MASP-2, a key serine protease in the lectin pathway of complement activation, resulting in excessive complement activation by potentiating MBL-dependent MASP-2 activation, and the deposition of MASP-2, C4b, activated C3 and C5b-9. Aggravated inflammatory lung injury was observed in mice infected with adenovirus expressing the N protein. Complement hyperactivation was also observed in SARS-CoV-2-infected patients. Either blocking the N protein:MASP-2 interaction, MASP-2 depletion or suppressing complement activation can significantly alleviate N protein-induced complement hyperactivation and lung injury in vitro and in vivo. Altogether, these data suggested that complement suppression may represent a novel therapeutic approach for pneumonia induced by these highly pathogenic coronaviruses.
Assuntos
COVID-19 , Lesão Pulmonar , Animais , COVID-19/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Inflamação/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Camundongos , SARS-CoV-2RESUMO
cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a Kd of 2.17 µM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted ß-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.
Assuntos
Diagnóstico por Imagem , Sistemas do Segundo Mensageiro , Animais , Corantes , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , CamundongosRESUMO
The interaction of the solo H3K79 methyltransferase DOT1-like (DOT1L) and its regulatory factor ALL1-fused gene from chromosome 10 protein (AF10) is crucial for the transcription of developmental genes such as HOXA in acute leukemia. The octapeptide motif and leucine zipper region of AF10 is responsible for binding DOT1L and catalyzing H3K79 monomethylation to demethylation. However, the characteristics of the mechanism between DOT1L and AF10 are not clear. Here, we present the crystal structures of coiled-coil regions of DOT1L-AF10 and AF10-inhibitory peptide, demonstrating the inhibitory peptide could form a compact complex with AF10 via a different recognition pattern. Furthermore, an inhibitory peptide with structure-based optimization is identified and decreases the HOXA gene expression in a human cell line. Our studies provide an innovative pharmacologic basis for therapeutic intervention in leukemia.
Assuntos
Antineoplásicos/farmacologia , Histona-Lisina N-Metiltransferase/química , Proteínas de Homeodomínio/biossíntese , Modelos Moleculares , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Regulação Neoplásica da Expressão Gênica/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Conformação ProteicaRESUMO
The worldwide epidemic of novel coronavirus disease (COVID-19) has led to a strong demand for highly efficient immunobinding to achieve rapid and accurate on-site detection of SARS-CoV-2 antibodies. However, hour-scale time-consumption is usually required to ensure the adequacy of immunobinding on expensive large instruments in hospitals, and the common false negative or positive results often occur in rapid on-site immunoassay (e.g. immunochromatography). We solved this dilemma by presenting a reciprocating-flowing immunobinding (RF-immunobinding) strategy. RF-immunobinding enabled the antibodies in fluid contacting with the corresponding immobilized antigens on substrate repeatedly during continuous reciprocating-flowing, to achieve adequate immunobinding within 60 s. This strategy was further developed into an immunoassay method for the serological detection of 13 suspected COVID-19 patients. We obtained a 100% true negative and true positive rate and a limit of quantification (LOQ) of 4.14 pg/mL. Our strategy also can be a potential support for other areas related to immunorecognition, such as proteomics, immunopharmacology and immunohistochemistry.
Assuntos
Teste Sorológico para COVID-19/instrumentação , COVID-19/diagnóstico , Dispositivos Lab-On-A-Chip , SARS-CoV-2/imunologia , Anticorpos Antivirais/sangue , Reações Antígeno-Anticorpo , Técnicas Biossensoriais/instrumentação , COVID-19/imunologia , COVID-19/virologia , Teste Sorológico para COVID-19/métodos , Ensaio de Imunoadsorção Enzimática/instrumentação , Desenho de Equipamento , Humanos , Proteínas Imobilizadas , PandemiasRESUMO
The development and utilization of inorganic material biosynthesis have evolved into single macromolecular systems. A putative cystathionine γ-lyase of bacteria Stenotrophomonas maltophilia (smCSE) is a newly identified biomolecule that enables the synthesis of nanomaterials. Due to the lack of structural information, the mechanism of smCSE biosynthesis remains unclear. Herein, we obtain two atomic-resolution smCSE-form X-ray structures and confirm that the conformational changes of Tyr108 and Lys206 within the enzyme active sites are critical for the protein-driven synthesis of metal sulfide quantum dots (QDs). The structural stability of tetramer and the specificity of surface amino acids are the basis for smCSE to synthesize quantum dots. The size of QD products can be regulated by predesigned amino acids and the morphology can be controlled through proteolytic treatments. The growth rate is enhanced by the stabilization of a flexible loop in the active site, as shown by the X-ray structure of the engineered protein which fused with a dodecapeptide. We further prove that the smCSE-driven route can be applied to the general synthesis of other metal sulfide nanoparticles. These results provide a better understanding of the mechanism of QD biosynthesis and a new perspective on the control of this biosynthesis by protein modification.
Assuntos
Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/ultraestrutura , Pontos Quânticos/química , Aminoácidos , Bactérias/metabolismo , Cistationina gama-Liase/química , Substâncias Macromoleculares , Metais , Nanoestruturas , Stenotrophomonas maltophilia/enzimologia , Stenotrophomonas maltophilia/metabolismo , Sulfetos/químicaRESUMO
Dysregulated immune cell responses have been linked to the severity of coronavirus disease 2019 (COVID-19), but the specific viral factors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were currently unknown. Herein, we reveal that the Immunoglobulin-like fold ectodomain of the viral protein SARS-CoV-2 ORF7a interacts with high efficiency to CD14+ monocytes in human peripheral blood, compared to pathogenic protein SARS-CoV ORF7a. The crystal structure of SARS-CoV-2 ORF7a at 2.2 Å resolution reveals three remarkable changes on the amphipathic side of the four-stranded ß-sheet, implying a potential functional interface of the viral protein. Importantly, SARS-CoV-2 ORF7a coincubation with CD14+ monocytes ex vivo triggered a decrease in HLA-DR/DP/DQ expression levels and upregulated significant production of proinflammatory cytokines, including IL-6, IL-1ß, IL-8, and TNF-α. Our work demonstrates that SARS-CoV-2 ORF7a is an immunomodulating factor for immune cell binding and triggers dramatic inflammatory responses, providing promising therapeutic drug targets for pandemic COVID-19.
RESUMO
Although human antibodies elicited by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-reactive antibodies. Herein, we isolate and profile a panel of 32 N protein-specific monoclonal antibodies (mAbs) from a quick recovery coronavirus disease-19 (COVID-19) convalescent patient who has dominant antibody responses to the SARS-CoV-2 N protein rather than to the SARS-CoV-2 spike (S) protein. The complex structure of the N protein RNA binding domain with the highest binding affinity mAb (nCoV396) reveals changes in the epitopes and antigen's allosteric regulation. Functionally, a virus-free complement hyperactivation analysis demonstrates that nCoV396 specifically compromises the N protein-induced complement hyperactivation, which is a risk factor for the morbidity and mortality of COVID-19 patients, thus laying the foundation for the identification of functional anti-N protein mAbs.
Assuntos
Anticorpos Antivirais/farmacologia , COVID-19/imunologia , Ativação do Complemento/efeitos dos fármacos , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Regulação Alostérica , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Convalescença , Proteínas do Nucleocapsídeo de Coronavírus/química , Cristalografia por Raios X , Epitopos , Humanos , Fosfoproteínas/química , Fosfoproteínas/imunologia , Conformação ProteicaRESUMO
ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.
Assuntos
COVID-19/imunologia , Quirópteros/imunologia , Imunidade/imunologia , Domínios de Imunoglobulina/imunologia , Proteínas Virais/imunologia , Animais , Sítios de Ligação/genética , COVID-19/virologia , Células Cultivadas , Quirópteros/genética , Quirópteros/metabolismo , Cristalografia por Raios X , Humanos , Imunidade/genética , Domínios de Imunoglobulina/genética , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Modelos Moleculares , Monócitos/imunologia , Monócitos/metabolismo , Mutação , Ligação Proteica , Especificidade da Espécie , Proteínas Virais/classificação , Proteínas Virais/genéticaRESUMO
B cell response plays a critical role against SARS-CoV-2 infection. However, little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection. Here, we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients, and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses. Via linking BCR to antigen specificity through sequencing (LIBRA-seq), we identified a distinct activated memory B cell subgroup (CD11chigh CD95high) had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells. Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection. The public antibody clonotypes were shared by distinct convalescent individuals. Moreover, several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain (RBD) or nucleoprotein (NP) via ELISA assay. Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro. Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.
Assuntos
Linfócitos B/imunologia , COVID-19/imunologia , Convalescença , Memória Imunológica , RNA-Seq , SARS-CoV-2/imunologia , Animais , Linfócitos B/patologia , COVID-19/genética , COVID-19/patologia , Linhagem Celular Tumoral , Separação Celular , Chlorocebus aethiops , Células HEK293 , Humanos , SARS-CoV-2/genética , Células VeroRESUMO
The emerging new lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have marked a new phase of coronavirus disease 2019 (COVID-19). Understanding the recognition mechanisms of potent neutralizing monoclonal antibodies (NAbs) against the spike protein is pivotal for developing new vaccines and antibody drugs. Here, we isolated several monoclonal antibodies (MAbs) against the SARS-CoV-2 spike protein receptor-binding domain (S-RBD) from the B cell receptor repertoires of a SARS-CoV-2 convalescent. Among these MAbs, the antibody nCoV617 demonstrates the most potent neutralizing activity against authentic SARS-CoV-2 infection, as well as prophylactic and therapeutic efficacies against the human angiotensin-converting enzyme 2 (ACE2) transgenic mouse model in vivo. The crystal structure of S-RBD in complex with nCoV617 reveals that nCoV617 mainly binds to the back of the "ridge" of RBD and shares limited binding residues with ACE2. Under the background of the S-trimer model, it potentially binds to both "up" and "down" conformations of S-RBD. In vitro mutagenesis assays show that mutant residues found in the emerging new lineage B.1.1.7 of SARS-CoV-2 do not affect nCoV617 binding to the S-RBD. These results provide a new human-sourced neutralizing antibody against the S-RBD and assist vaccine development. IMPORTANCE COVID-19 is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID-19 pandemic has posed a serious threat to global health and the economy, so it is necessary to find safe and effective antibody drugs and treatments. The receptor-binding domain (RBD) in the SARS-CoV-2 spike protein is responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor. It contains a variety of dominant neutralizing epitopes and is an important antigen for the development of new coronavirus antibodies. The significance of our research lies in the determination of new epitopes, the discovery of antibodies against RBD, and the evaluation of the antibodies' neutralizing effect. The identified antibodies here may be drug candidates for the development of clinical interventions for SARS-CoV-2.