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1.
Proc Natl Acad Sci U S A ; 115(22): E5135-E5143, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29760102

RESUMO

Porcine deltacoronavirus (PDCoV), identified in 2012, is a common enteropathogen of swine with worldwide distribution. The source and evolutionary history of this virus is, however, unknown. PDCoV belongs to the Deltacoronavirus genus that comprises predominantly avian CoV. Phylogenetic analysis suggests that PDCoV originated relatively recently from a host-switching event between birds and mammals. Insight into receptor engagement by PDCoV may shed light into such an exceptional phenomenon. Here we report that PDCoV employs host aminopeptidase N (APN) as an entry receptor and interacts with APN via domain B of its spike (S) protein. Infection of porcine cells with PDCoV was drastically reduced by APN knockout and rescued after reconstitution of APN expression. In addition, we observed that PDCoV efficiently infects cells of unusual broad species range, including human and chicken. Accordingly, PDCoV S was found to target the phylogenetically conserved catalytic domain of APN. Moreover, transient expression of porcine, feline, human, and chicken APN renders cells susceptible to PDCoV infection. Binding of PDCoV to an interspecies conserved site on APN may facilitate direct transmission of PDCoV to nonreservoir species, including humans, potentially reflecting the mechanism that enabled a virus, ancestral to PDCoV, to breach the species barrier between birds and mammals. The APN cell surface protein is also used by several members of the Alphacoronavirus genus. Hence, our data constitute the second identification of CoVs from different genera that use the same receptor, implying that CoV receptor selection is subjected to specific restrictions that are still poorly understood.


Assuntos
Doenças Transmissíveis Emergentes , Infecções por Coronavirus , Coronavirus/fisiologia , Coronavirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Animais , Anticorpos Antivirais/imunologia , Antígenos CD13/metabolismo , Gatos , Linhagem Celular , Galinhas , Chlorocebus aethiops , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/veterinária , Doenças Transmissíveis Emergentes/virologia , Coronavirus/imunologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Cães , Especificidade de Hospedeiro , Humanos , Células Madin Darby de Rim Canino , Camundongos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Suínos , Células Vero , Zoonoses/transmissão , Zoonoses/virologia
2.
Emerg Infect Dis ; 26(5): 1024-1027, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32150528

RESUMO

We developed and validated 2 species-independent protein-based assays to detect Middle East respiratory syndrome coronavirus functional antibodies that can block virus receptor-binding or sialic acid-attachment. Antibody levels measured in both assays correlated strongly with virus-neutralizing antibody titers, proving their use for serologic confirmatory diagnosis of Middle East respiratory syndrome.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Testes Sorológicos/métodos , Anticorpos Neutralizantes/sangue , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Especificidade da Espécie
3.
Proc Natl Acad Sci U S A ; 114(40): E8508-E8517, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28923942

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1A through S1D Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1B We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1A When multivalently displayed on nanoparticles, S1 or S1A bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5-N-glycolylation and (7,)9-O-acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Polissacarídeos/metabolismo , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Camelus , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Humanos , Mucinas , Glicoproteína da Espícula de Coronavírus/genética , Ligação Viral
4.
Emerg Infect Dis ; 25(10): 1868-1877, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31423970

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in humans can cause asymptomatic to fatal lower respiratory lung disease. Despite posing a probable risk for virus transmission, asymptomatic to mild infections can go unnoticed; a lack of seroconversion among some PCR-confirmed cases has been reported. We found that a MERS-CoV spike S1 protein-based ELISA, routinely used in surveillance studies, showed low sensitivity in detecting infections among PCR-confirmed patients with mild clinical symptoms and cross-reactivity of human coronavirus OC43-positive serum samples. Using in-house S1 ELISA and protein microarray, we demonstrate that most PCR-confirmed MERS-CoV case-patients with mild infections seroconverted; nonetheless, some of these samples did not have detectable levels of virus-neutralizing antibodies. The use of a sensitive and specific serologic S1-based assay can be instrumental in the accurate estimation of MERS-CoV prevalence.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Imunidade Humoral/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Fatores de Tempo
5.
J Virol ; 90(13): 5965-5977, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27099320

RESUMO

UNLABELLED: Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE: RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Epitopos/imunologia , Imunidade Inata , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Animais , Anticorpos Antivirais/imunologia , Formaldeído , Humanos , Epitopos Imunodominantes/sangue , Epitopos Imunodominantes/imunologia , Ratos , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/efeitos adversos , Vacinas contra Vírus Sincicial Respiratório/química , Vírus Sincicial Respiratório Humano/química , Sigmodontinae , Vacinação/efeitos adversos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Proteínas Virais/imunologia
6.
Plant J ; 61(2): 249-58, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19843314

RESUMO

Using a proteomics approach, a PP2C-type phosphatase (renamed PIA1, for PP2C induced by AvrRpm1) was identified that accumulates following infection by Pseudomonas syringae expressing the type III effector AvrRpm1, and subsequent activation of the corresponding plant NB-LRR disease resistance protein RPM1. No accumulation of PIA1 protein was seen following infection with P. syringae expressing AvrB, another type III effector that also activates RPM1, although PIA transcripts were observed. Accordingly, mutation of PIA1 resulted in enhanced RPM1 function in response to P. syringae pathover tomato (Pto) DC3000 (avrRpm1) but not to Pto DC3000 (avrB). Thus, PIA1 is a protein marker that distinguishes AvrRpm1- and AvrB-dependent activation of RPM1. AvrRpm1-induced expression of the pathogenesis-related genes PR1, PR2 and PR3, and salicylic acid accumulation were reduced in two pia1 mutants. By contrast, expression of other defense-related genes, including PR5 and PDF1.2 (plant defensin), was elevated in unchallenged pia1 mutants. Hence, PIA1 is required for AvrRpm1-induced responses, and confers dual (both positive and negative) regulation of defense gene expression.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Bactérias/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Pseudomonas syringae/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Western Blotting , Eletroforese em Gel Bidimensional , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Espectrometria de Massas , Mutação , Fosfoproteínas Fosfatases/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Proteína Fosfatase 2C , Proteômica , Pseudomonas syringae/genética , Pseudomonas syringae/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
J Virol ; 84(18): 9625-31, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20631148

RESUMO

We have demonstrated that influenza A virus (IAV) RNA synthesis depends on the ubiquitin-proteasome system. IAV replication was reduced both by proteasome inhibitors and in E36ts20 cells, which contain the thermolabile ubiquitin-activating enzyme E1. While virus entry was not affected in E36ts20 cells, the proteasome inhibitor MG132 retained viral particles in the cytoplasm. Addition-removal experiments of MG132 in combination with bafilomycin A1, a well-established inhibitor of IAV entry and fusion, showed that MG132 affected IAV infection at a postfusion step. This was confirmed by the lack of inhibition of IAV entry by proteasome inhibitors in a virus-like particle fusion assay.


Assuntos
Vírus da Influenza A/fisiologia , Inibidores de Proteassoma , Ubiquitina/antagonistas & inibidores , Replicação Viral , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Inibidores Enzimáticos/farmacologia , Humanos , Leupeptinas/farmacologia , Macrolídeos/farmacologia
8.
Nat Commun ; 12(1): 1715, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731724

RESUMO

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Betacoronavirus/imunologia , Epitopos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Betacoronavirus/classificação , Camelus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/genética , Humanos , Camundongos , Conformação Proteica , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
9.
Emerg Microbes Infect ; 9(1): 1080-1091, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32471334

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.


Assuntos
Formação de Anticorpos , Apresentação de Antígeno , Infecções por Coronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Sítios de Ligação , Infecções por Coronavirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Células HEK293 , Humanos , Imunogenicidade da Vacina , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Testes de Neutralização , Ligação Proteica , Domínios Proteicos , Coelhos , Glicoproteína da Espícula de Coronavírus/biossíntese , Replicação Viral
10.
Proteomics ; 9(1): 138-47, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19053141

RESUMO

Transgenic Arabidopsis conditionally expressing the bacterial avrRpm1 type III effector under the control of a dexamethasone-responsive promoter were used for proteomics studies. This model system permits study of an individual effector without interference from additional bacterial components. Coupling of different prefractionation approaches to high resolution 2-DE facilitated the discovery of low abundance proteins - enabling the identification of proteins that have escaped detection in similar experiments. A total of 34 differentially regulated protein spots were identified. Four of these (a remorin, a protein phosphatase 2C (PP2C), an RNA-binding protein, and a C2-domain-containing protein) are potentially early signaling components in the interaction between AvrRpm1 and the cognate disease resistance gene product, resistance to Pseudomonas syringae pv. maculicola 1 (RPM1). For the remorin and RNA-binding protein, involvement of PTM and post-transcriptional regulation are implicated, respectively.


Assuntos
Proteínas de Arabidopsis/isolamento & purificação , Arabidopsis/química , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Proteômica/métodos , Arabidopsis/imunologia , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Imunidade Inata , Microssomos/química , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/fisiologia , Ribulose-Bifosfato Carboxilase/química
11.
Emerg Microbes Infect ; 8(1): 516-530, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30938227

RESUMO

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Imunização Passiva , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Domínios Proteicos , Receptores Virais/genética , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/genética
12.
Sci Adv ; 4(8): eaas9667, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30101189

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries-apart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV-infected dromedaries, we identified several MERS-CoV-specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.


Assuntos
Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Infecções por Coronavirus/prevenção & controle , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Camelus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Camundongos , Testes de Neutralização , Ligação Proteica , Anticorpos de Domínio Único
13.
PLoS One ; 12(1): e0170877, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28135305

RESUMO

Respiratory syncytial virus (RSV) is the leading cause for respiratory illness that requires hospitalization in infancy. High levels of maternal antibodies can protect against RSV infection. However, RSV-infected infants can suffer from severe disease symptoms even in the presence of high levels of RSV-specific antibodies. This study analyzes several serological characteristics to explore potential deficiencies or surpluses of antibodies that could relate to severe disease symptoms. We compare serum antibodies from hospitalized patients who suffered severe symptoms as well as uninfected infants. Disease severity markers were oxygen therapy, tachypnea, oxygen saturation, admission to the intensive care unit and duration of hospitalization. Antibodies against RSV G protein and a prefusion F epitope correlated with in vitro neutralization. Avidity of RSV-specific IgG antibodies was lower in RSV-infected infants compared to uninfected controls. Severe disease symptoms were unrelated to RSV-specific IgG antibody titers, avidity of RSV-IgG, virus neutralization capacity or titers against pre- and postfusion F or G protein ectodomains and the prefusion F antigenic site Ø. In conclusion, the detailed serological characterization did not indicate dysfunctional or epitope-skewed composition of serum antibodies in hospitalized RSV-infected infants suffering from severe disease symptoms. It remains unclear, whether specific antibody fractions could diminish disease symptoms.


Assuntos
Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Hospitalização , Infecções por Vírus Respiratório Sincicial/sangue , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Doença Aguda , Afinidade de Anticorpos/imunologia , Antígenos Virais/imunologia , Epitopos/imunologia , Feminino , Glicoproteínas/imunologia , Humanos , Imunoglobulina G/sangue , Lactente , Masculino , Testes de Neutralização , Índice de Gravidade de Doença
14.
Antiviral Res ; 122: 46-50, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26259810

RESUMO

Respiratory syncytial virus (RSV) is the major cause for respiratory tract disease in infants and young children. Currently, no licensed vaccine or a selective antiviral drug against RSV infections are available. Here, we describe a structure-based drug design approach that led to the synthesis of a novel series of zinc-ejecting compounds active against RSV replication. 30 compounds, sharing a common dithiocarbamate moiety, were designed and prepared to target the zinc finger motif of the M2-1 protein. A library of ∼ 12,000 small fragments was docked to explore the area surrounding the zinc ion. Among these, seven ligands were selected and used for the preparation of the new derivatives. The results reported here may help the development of a lead compound for the treatment of RSV infections.


Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Simulação por Computador , Desenho de Fármacos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Zinco/metabolismo , Antivirais/química , Sítios de Ligação , Linhagem Celular Tumoral , Desenho Assistido por Computador , Etilenobis (ditiocarbamatos)/química , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Zinco/química , Dedos de Zinco/efeitos dos fármacos
15.
PLoS One ; 10(6): e0130829, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26107504

RESUMO

The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble, trimeric, non-cleaved RSV F protein, produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence, is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast, a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø, but did not expose postfusion-specific antigenic site I, in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile, alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB, the exposure of postfusion-specific antigenic site I was not prevented. In conclusion, proteins that are not able to form the 6HB, due to mutation of HRB, may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted, however, in a protein with prefusion-like characteristics, suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.


Assuntos
Anticorpos Antivirais/farmacologia , Células Epiteliais/metabolismo , Mucosa Respiratória/metabolismo , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genética , Anticorpos Neutralizantes/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Células Epiteliais/patologia , Células Epiteliais/virologia , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Virais de Fusão/antagonistas & inibidores , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo
16.
J Biomol Tech ; 15(2): 97-106, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15190082

RESUMO

Comparative proteomics of leaves, flowers, and glands of Cannabis sativa have been used to identify specific tissue-expressed proteins. These tissues have significantly different levels of cannabinoids. Cannabinoids accumulate primarily in the glands but can also be found in flowers and leaves. Proteins extracted from glands, flowers, and leaves were separated using two-dimensional gel electrophoresis. Over 800 protein spots were reproducibly resolved in the two-dimensional gels from leaves and flowers. The patterns of the gels were different and little correlation among the proteins could be observed. Some proteins that were only expressed in flowers were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprint database searching. Flower and gland proteomes were also compared, with the finding that less then half of the proteins expressed in flowers were also expressed in glands. Some selected gland protein spots were identified: F1D9.26-unknown prot. (Arabidopsis thaliana), phospholipase D beta 1 isoform 1a (Gossypium hirsutum), and PG1 (Hordeum vulgare). Western blotting was employed to identify a polyketide synthase, an enzyme believed to be involved in cannabinoid biosynthesis, resulting in detection of a single protein.


Assuntos
Cannabis/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/análise , Western Blotting , Canabinoides/metabolismo , Cannabis/enzimologia , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Flores/metabolismo , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Ponto Isoelétrico , Peso Molecular , Mapeamento de Peptídeos , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Distribuição Tecidual
17.
PLoS One ; 8(8): e71072, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23951084

RESUMO

Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease in infants and the elderly. Currently, no licensed vaccine against RSV is available. Here we describe the development of a safe and effective intranasal subunit vaccine that is based on recombinant fusion (F) protein bound to the surface of immunostimulatory bacterium-like particles (BLPs) derived from the food-grade bacterium Lactococcus lactis. Different variants of F were analyzed with respect to their conformation and reactivity with neutralizing antibodies, assuming that F proteins mimicking the metastable prefusion form of RSV F expose a more extensive and relevant epitope repertoire than F proteins corresponding to the postfusion structure. Our results indicate that the recombinant soluble ectodomain of RSV F readily adopts a postfusion conformation, generation of which cannot be prevented by C-terminal addition of a trimerization motif, but whose formation is prevented by mutation of the two furin cleavage sites in F. While the putative postfusion form of F is recognized well by the monoclonal antibody Palivizumab, this is much less so for the more potently neutralizing, prefusion-specific antibodies D25 and AM22. Both addition of the trimerization motif and mutation of the furin cleavage sites increased the reactivity of F with D25 and AM22, with the highest reactivity being observed for F proteins in which both these features were combined. Intranasal vaccination of mice or cotton rats with BLPs loaded with this latter prefusion-like F protein (BLP-F), resulted in the potent induction of F-specific immunoglobulins and in significantly decreased virus titers in the lungs upon RSV challenge. Moreover, and in contrast to animals vaccinated with formalin-inactivated RSV, animals that received BLP-F exhibited high levels of F-specific secretory IgA in the nose and RSV-neutralizing antibodies in sera, but did not show symptoms of enhanced disease after challenge with RSV.


Assuntos
Proteínas Recombinantes de Fusão/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Administração Intranasal , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Humanos , Lactococcus lactis/imunologia , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Proteínas Recombinantes de Fusão/genética , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/efeitos adversos , Vírus Sincicial Respiratório Humano/genética , Sigmodontinae , Vacinação , Proteínas Virais de Fusão/genética
18.
PLoS One ; 7(10): e47529, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23071819

RESUMO

Influenza A virus (IAV) contains a segmented negative-strand RNA genome. How IAV balances the replication and transcription of its multiple genome segments is not understood. We developed a dual competition assay based on the co-transfection of firefly or Gaussia luciferase-encoding genome segments together with plasmids encoding IAV polymerase subunits and nucleoprotein. At limiting amounts of polymerase subunits, expression of the firefly luciferase segment was negatively affected by the presence of its Gaussia luciferase counterpart, indicative of competition between reporter genome segments. This competition could be relieved by increasing or decreasing the relative amounts of firefly or Gaussia reporter segment, respectively. The balance between the luciferase expression levels was also affected by the identity of the untranslated regions (UTRs) as well as segment length. In general it appeared that genome segments displaying inherent higher expression levels were more efficient competitors of another segment. When natural genome segments were tested for their ability to suppress reporter gene expression, shorter genome segments generally reduced firefly luciferase expression to a larger extent, with the M and NS segments having the largest effect. The balance between different reporter segments was most dramatically affected by the introduction of UTR panhandle-stabilizing mutations. Furthermore, only reporter genome segments carrying these mutations were able to efficiently compete with the natural genome segments in infected cells. Our data indicate that IAV genome segments compete for available polymerases. Competition is affected by segment length, coding region, and UTRs. This competition is probably most apparent early during infection, when limiting amounts of polymerases are present, and may contribute to the regulation of segment-specific replication and transcription.


Assuntos
Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Vírus da Influenza A/genética , Luciferases/genética , RNA não Traduzido/genética , Animais , Copépodes/enzimologia , Primers do DNA/genética , Vaga-Lumes/enzimologia , Genes Reporter/genética , Mutação/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção
19.
J Asian Nat Prod Res ; 4(1): 63-7, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11991194

RESUMO

Cell suspension cultures of Solanum mammosum cultivated in modified Murashige & Skoog media could synthesize o-aminobenzoic acid-7-O-beta-D-(beta-1,6-O-D-glucopyranosyl)-glucopyranosyl ester from o-amino benzoic acid with a yield of about 20% dry weight in 7 days. The maximum production of o-aminobenzoic acid-7-O-beta-D-(beta-1,6-O-D-glucopyranosyl)-glucopyranosyl ester was 31.8% on dry weight basis.


Assuntos
Aminobenzoatos/química , Aminobenzoatos/metabolismo , Dissacarídeos/química , Solanaceae/química , Biotransformação , Técnicas de Cultura de Células , Ésteres/química
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