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1.
Cell Host Microbe ; 27(3): 441-453.e7, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32004500

RESUMO

Rabies virus (RABV), the etiological agent for the lethal disease of rabies, is a deadly zoonotic pathogen. The RABV glycoprotein (RABV-G) is a key factor mediating virus entry and the major target of neutralizing antibodies. Here, we report the crystal structures of RABV-G solved in the free form at ∼pH-8.0 and in the complex form with a neutralizing antibody 523-11 at ∼pH-6.5, respectively. RABV-G has three domains, and the basic-to-acidic pH change results in large domain re-orientations and concomitant domain-linker re-constructions, switching it from a bent hairpin conformation into an extended conformation. During such low-pH-induced structural transitions, residues located in the domain-linker are found to play important roles in glycoprotein-mediated membrane fusion. Finally, the antibody interacts with RABV-G mainly through its heavy chain and binds to a bipartite conformational epitope in the viral protein for neutralization. These structures provide valuable information for vaccine and drug design.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Antígenos Virais/química , Concentração de Íons de Hidrogênio , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Fusão Celular , Linhagem Celular , Cricetinae , Multimerização Proteica , Estrutura Terciária de Proteína , Vírus da Raiva
2.
Ecotoxicology ; 29(2): 156-162, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31927676

RESUMO

Nanomaterials have revolutionized many scientific fields and are widely applied to address environmental problems and to develop novel health care strategies. However, their mechanism of action is still poorly understood. Several nanomaterials for medical applications are based on quantum dots (QDs). Despite their amazing physico-chemical properties, quantum dots display significant adverse effects. In the present study, the effects of QDs on the motor nervous system of nematodes Caenorhabditis elegans have been investigated as a non-mammalian alternative model. We also explored the possibility of modifying the toxicity of QDs by coating with a cell-penetrating peptide gH625 and thus we analysed the effects determined by QDs-gH625 complexes on the nematodes. With this work, we have demonstrated, by in vivo experiments, that the peptide gH625 is able to reduce the side effects of metallic nanoparticle making them more suitable for medical applications.


Assuntos
Caenorhabditis elegans/fisiologia , Estresse Oxidativo/fisiologia , Pontos Quânticos , Animais , Modelos Biológicos , Peptídeos/química , Proteínas do Envelope Viral/química
3.
Emerg Microbes Infect ; 8(1): 1584-1592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682199

RESUMO

The genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.


Assuntos
Variação Antigênica , Vírus da Raiva/imunologia , Raiva/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Feminino , Cobaias , Humanos , Testes de Neutralização , Raiva/imunologia , Raiva/prevenção & controle , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/genética , Vacinas Antirrábicas/imunologia , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
4.
PLoS Pathog ; 15(9): e1007996, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31536610

RESUMO

The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.


Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/genética , Linhagem Celular , Microscopia Crioeletrônica , Vírus da Dengue/ultraestrutura , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência de Aminoácidos , Sorogrupo , Temperatura Ambiente , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
5.
Int J Mol Sci ; 20(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370181

RESUMO

The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.


Assuntos
Fragmentos de Peptídeos/química , Polissacarídeos/química , Proteínas do Envelope Viral/química , Sulfato de Amônio/química , Animais , Células CHO , Sequência de Carboidratos , Precipitação Química , Cromatografia em Gel/métodos , Cricetulus , Galactose/química , Expressão Gênica , Glicosilação , Humanos , Ácido N-Acetilneuramínico/química , Fragmentos de Peptídeos/isolamento & purificação , Células Vegetais/metabolismo , Polissacarídeos/isolamento & purificação , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
6.
Nanoscale ; 11(35): 16368-16376, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31436278

RESUMO

Antiviral agents work by stopping or intervening the virus replication. Virus replication is a fast and multi-step process while effective antiviral intervention requires agents to interact with the protein coat, genetic RNA/DNA or both during virus replication. Thus, quantifying these interactions at the molecular level, although it is quite challenging, is very important for an understanding of the underlying molecular mechanism of antiviral intervention. Here, at the single molecule level, we employ single molecule force spectroscopy (SMFS) in combination with AFM imaging and choose tobacco mosaic virus (TMV)/tannin as a model system of tubular virus to directly study how the inhibitor influences the interactions of RNA and coat protein. We illustrated the antiviral mechanism of tannin during the three main stages of TMV infection, i.e., before the entry of cells, the disassembly of genetic RNA and reassembly of genetic RNA, respectively. Our SMFS results show that tannin and its derivatives can stabilize the TMV complex by enhancing the interactions between RNA and coat protein via weak interactions, such as hydrogen bonding and hydrophobic interactions. In addition, the stabilization effect showed molecular weight dependence, i.e., for higher molecular weight tannin the stabilization occurs after genetic RNA gets partially disassembled from the protein coat, while the lower molecular weight tannin hydrolyte starts experiencing the stabilization effect before the RNA disassembly. Furthermore, the cycling stretching-relaxation experiments in the presence/absence of tannin proved that tannin can prevent the assembling of RNA and coat protein. In addition, the AFM imaging results demonstrate that tannin can cause the aggregation of TMV particles in a concentration-dependent manner; a higher concentration of tannin will cause more severe aggregations. These results deepen our understanding of the antiviral mechanism of tannin and its derivatives, which facilitate the rational design of efficient agents for antiviral therapy.


Assuntos
Antivirais/química , Microscopia de Força Atômica , RNA Viral/química , Taninos/química , Vírus do Mosaico do Tabaco , Proteínas do Envelope Viral/química , Interações Hidrofóbicas e Hidrofílicas , Vírus do Mosaico do Tabaco/química , Vírus do Mosaico do Tabaco/ultraestrutura
7.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261659

RESUMO

Congenital human cytomegalovirus (HCMV) infection and HCMV infection of immunosuppressed patients cause significant morbidity and mortality, and vaccine development against HCMV is a major public health priority. HCMV envelope glycoproteins gB, gH, and gL, which constitute the core fusion machinery, play critical roles in HCMV fusion and entry into host cells. HCMV gB and gH/gL have been reported to elicit potent neutralizing antibodies. Recently, the gB/gH/gL complex was identified in the envelope of HCMV virions, and 16-50% of the total gH/gL bound to gB, forming the gB/gH/gL complex. These findings make the gB/gH/gL a unique HCMV vaccine candidate. We previously reported the production of HCMV trimeric gB and gH/gL heterodimers, and immunization with a combination of trimeric gB and gH/gL heterodimers elicited strong synergistic HCMV-neutralizing activity. To further improve the immunogenicity of gH/gL, we produced trimeric gH/gL. Rabbits immunized with HCMV trimeric gH/gL induced up to 38-fold higher serum titers of gH/gL-specific IgG relative to HCMV monomeric gH/gL, and elicited ~10-fold higher titers of complement-dependent and complement-independent HCMV-neutralizing activity for both epithelial cells and fibroblasts. HCMV trimeric gH/gL in combination with HCMV trimeric gB would be a novel promising HCMV vaccine candidate that could induce highly potent neutralizing activities.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra Citomegalovirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Coelhos , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
8.
PLoS One ; 14(7): e0219995, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31329627

RESUMO

Prion diseases are members of neurodegenerative protein misfolding diseases (NPMDs) that include Alzheimer's, Parkinson's and Huntington diseases, amyotrophic lateral sclerosis, tauopathies, traumatic brain injuries, and chronic traumatic encephalopathies. No known therapeutics extend survival or improve quality of life of humans afflicted with prion disease. We and others developed a new approach to NPMD therapy based on reducing the amount of the normal, host-encoded protein available as substrate for misfolding into pathologic forms, using RNA interference, a catabolic pathway that decreases levels of mRNA encoding a particular protein. We developed a therapeutic delivery system consisting of small interfering RNA (siRNA) complexed to liposomes and addressed to the central nervous system using a targeting peptide derived from rabies virus glycoprotein. These liposome-siRNA-peptide complexes (LSPCs) cross the blood-brain barrier and deliver PrP siRNA to neuronal cells to decrease expression of the normal cellular prion protein, PrPC, which acts as a substrate for prion replication. Here we show that LSPCs can extend survival and improve behavior of prion-infected mice that remain immunotolerant to treatment. LSPC treatment may be a viable therapy for prion and other NPMDs that can improve the quality of life of patients at terminal disease stages.


Assuntos
Lipossomos/metabolismo , Proteínas PrPC/genética , Doenças Priônicas/terapia , Terapêutica com RNAi/métodos , Animais , Antígenos Virais/química , Antígenos Virais/metabolismo , Barreira Hematoencefálica/metabolismo , Feminino , Lipossomos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPC/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
9.
Int J Mol Sci ; 20(14)2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311082

RESUMO

Dengue, one of the most prevalent illnesses caused by dengue viruses that are members of the genus Flavivirus, is a significant global health problem. However, similar clinical symptoms and high antigenic homologies with other Flaviviruses in the endemic area pose difficulties for differential diagnosis of dengue from other arbovirus infections. Here, we investigated four types of recombinant envelope protein domain III (DV-rED III) derived from four dengue virus (DENV) serotypes for diagnostic potential in detecting IgM in acute phase (mainly 2-3 days after onset of fever). Each independent DV-1, -3, and -4-rED III-ELISA showed less than 60% sensitivity, but the combined results of DV-1, -3, and -4-rED III-ELISA led to sensitivity of 81.82% (18/22) (95% CI, 59.72 to 94.81) and 100% specificity (46/46) (95% CI, 92.29 to 100.00) as each antigen compensated the other antigen-derived negative result. In conclusion, the independent combination of data derived from each recombinant antigen (DV1-, DV3-, and DV4-rED III) showed comparable efficacy for the detection of IgM in patients with acute-phase dengue infection.


Assuntos
Vírus da Dengue/imunologia , Dengue/diagnóstico , Testes Sorológicos/métodos , Proteínas do Envelope Viral/imunologia , Adulto , Anticorpos Antivirais/imunologia , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/genética , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina M/imunologia , Masculino , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos/normas , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética
10.
Biomolecules ; 9(6)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242697

RESUMO

In practice and despite Anfinsen's dogma, the refolding of recombinant multiple SS-bonded proteins is famously difficult because misfolded species with non-native SS-bonds appear upon the oxidization of their cysteine residues. On the other hand, single SS-bond proteins are thought to be simple to refold because their cysteines have only one SS-bond partner. Here, we report that dengue 4 envelope protein domain 3 (DEN4 ED3), a single SS-bonded protein can be irreversibly trapped into a misfolded species through the formation of its sole intramolecular SS-bond. The misfolded species had a much lower solubility than the native one at pHs higher than about 7, and circular dichroism measurements clearly indicated that its secondary structure content was different from the native species. Furthermore, the peaks in the Heteronuclear Single Quantum Correlation spectroscopy (HSQC) spectrum of DEN4 ED3 from the supernatant fraction were sharp and well dispersed, reflecting the beta-sheeted native structure, whereas the spectrum of the precipitated fraction showed broad signals clustered near its center suggesting no or little structure and a strong tendency to aggregate. The two species had distinct biophysical properties and could interconvert into each other only by cleaving and reforming the SS-bond, strongly suggesting that they are topologically different. This phenomenon can potentially happen with any single SS-bonded protein, and our observation emphasizes the need for assessing the conformation and biophysical properties of bacterially produced therapeutic proteins in addition to their chemical purities.


Assuntos
Dissulfetos/química , Dobramento de Proteína , Proteínas do Envelope Viral/química , Fenômenos Biofísicos , Precipitação Química , Conformação Proteica , Solubilidade
11.
Int J Pharm ; 567: 118449, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31226473

RESUMO

In the present study, we have prepared microRNA(miR)-124-loaded Rabies virus glycoprotein (RVG)29 surface-conjugated polymeric nanoparticles (NPs) to improve neuroinflammation in Parkinson's disease (PD). We hypothesize that an increase in the intracellular concentration of miR-124 will result in a better prognosis for Parkinson's disease. Minimal toxicity for the RVG29 NPs was observed at concentrations <100 µg/mL, while the cell viability of cells treated with blank NPs at concentrations of 200 µg/mL markedly decreased, indicating the safety of the carrier system. Results showed that mRNA levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-6, significantly increased upon lipopolysaccharide (LPS) administration. However, the mRNA levels of these cytokines reflected those of the miR-NPs-treated control group, indicating the influence of miR-124 exposure. After transfection with miR-NPs, levels of pro-inflammatory cytokines and neuroprotective molecules were reduced and increased, respectively. Administration of LPS significantly increased the levels of mitogen activated protein kinase kinase kinase (MEKK)3 and P-P65 levels, while transfection with miR-NPs significantly reduced the expression of both MEKK3 and P-P65, reflecting that of the control. This research has revealed that miR-124 could target both the MEKK3 and nuclear factor kappa light chain enhancer of activated B cell (NF-Kb) pathways, while also reducing inflammatory cytokine levels. In addition, a 3-fold decrease in apoptosis was observed in miR-NP transfected cells. The exogenous delivery of miR-NPs significantly downregulated MEKK3 expression in animal studies, as outlined by immunohistochemical staining (IHC). Overall, miR-NPs have the potential to inhibit pro-inflammatory signaling and enhance neuroprotection in PD.


Assuntos
MicroRNAs/administração & dosagem , Nanopartículas/administração & dosagem , Transtornos Parkinsonianos/terapia , Fragmentos de Peptídeos/administração & dosagem , Proteínas do Envelope Viral/administração & dosagem , Animais , Liberação Controlada de Fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/química , Nanopartículas/química , Fragmentos de Peptídeos/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas do Envelope Viral/química
12.
Virol Sin ; 34(5): 521-537, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31161554

RESUMO

The phosphatidylserine-specific phospholipase A1 (PLA1A) is an essential host factor in hepatitis C virus (HCV) assembly. In this study, we mapped the E2, NS2 and NS5A involved in PLA1A interaction to their lumenal domains and membranous parts, through which they form oligomeric protein complexes to participate in HCV assembly. Multiple regions of PLA1A were involved in their interaction and complex formation. Furthermore, the results represented structures with PLA1A and E2 in closer proximity than NS2 and NS5A, and strongly suggest PLA1A-E2's physical interaction in cells. Meanwhile, we mapped the NS5A sequence which participated in PLA1A interaction with the C-terminus of domain 1. Interestingly, these amino acids in the sequence are also essential for viral RNA replication. Further experiments revealed that these four proteins interact with each other. Moreover, PLA1A expression levels were elevated in livers from HCV-infected patients. In conclusion, we exposed the structural determinants of PLA1A, E2, NS2 and NS5A proteins which were important for HCV assembly and provided a detailed characterization of PLA1A in HCV assembly.


Assuntos
Hepacivirus/fisiologia , Fosfatidilserinas/química , Fosfolipases A1/química , Proteínas do Envelope Viral/química , Montagem de Vírus , Linhagem Celular Tumoral , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Fígado/virologia , Fosfolipases A1/genética , RNA Viral/metabolismo , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
13.
PLoS Pathog ; 15(5): e1007759, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116791

RESUMO

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.


Assuntos
Hepacivirus/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Anticorpos Neutralizantes/metabolismo , Células HEK293 , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Conformação Proteica , Multimerização Proteica , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus
14.
Soft Matter ; 15(22): 4525-4540, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31099376

RESUMO

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.


Assuntos
HIV-1/química , Manose/química , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/química , Proteínas do Envelope Viral/química , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Manose/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Nanopartículas Metálicas/química , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
15.
Virol J ; 16(1): 69, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133031

RESUMO

BACKGROUND: Coronaviruses (CoVs) primarily cause enzootic infections in birds and mammals but, in the last few decades, have shown to be capable of infecting humans as well. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and, more recently, Middle-East respiratory syndrome (MERS) has demonstrated the lethality of CoVs when they cross the species barrier and infect humans. A renewed interest in coronaviral research has led to the discovery of several novel human CoVs and since then much progress has been made in understanding the CoV life cycle. The CoV envelope (E) protein is a small, integral membrane protein involved in several aspects of the virus' life cycle, such as assembly, budding, envelope formation, and pathogenesis. Recent studies have expanded on its structural motifs and topology, its functions as an ion-channelling viroporin, and its interactions with both other CoV proteins and host cell proteins. MAIN BODY: This review aims to establish the current knowledge on CoV E by highlighting the recent progress that has been made and comparing it to previous knowledge. It also compares E to other viral proteins of a similar nature to speculate the relevance of these new findings. Good progress has been made but much still remains unknown and this review has identified some gaps in the current knowledge and made suggestions for consideration in future research. CONCLUSIONS: The most progress has been made on SARS-CoV E, highlighting specific structural requirements for its functions in the CoV life cycle as well as mechanisms behind its pathogenesis. Data shows that E is involved in critical aspects of the viral life cycle and that CoVs lacking E make promising vaccine candidates. The high mortality rate of certain CoVs, along with their ease of transmission, underpins the need for more research into CoV molecular biology which can aid in the production of effective anti-coronaviral agents for both human CoVs and enzootic CoVs.


Assuntos
Coronavirus/química , Proteínas do Envelope Viral/química , Animais , Coronavirus/genética , Coronavirus/patogenicidade , Infecções por Coronavirus/virologia , Humanos , Vírus da SARS/química , Vírus da SARS/genética , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/genética , Zoonoses/transmissão , Zoonoses/virologia
16.
Am J Trop Med Hyg ; 101(1): 233-236, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31115302

RESUMO

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern because of its recent geographic expansion. Although commercial vaccines are available and used in some endemic countries, JEV continues to cause illness, with more than 60,000 cases reported annually. To develop a reproducible positive control antibody useable in diagnosis of JEV infections, murine hybridomas were developed from mice inoculated with a combination of IXIARO JEV vaccine and JEV domain III of the envelope protein (E-DIII). Monoclonal antibodies (MAbs) were characterized for their ability to neutralize virus in vitro. Monoclonal antibody 17BD3-2 was found to be JEV specific and highly neutralizing, with a plaque reduction neutralization test (PRNT)90 endpoint titer of 1.25 µg/mL. The functional epitopes were mapped using virus neutralization escape variants to amino acid residues S309, K312, and G333 in E-DIII. This MAb may be substituted for human immune sera used as a positive control in PRNT for distribution to public health laboratories worldwide in potential future outbreaks of JEV.


Assuntos
Anticorpos Monoclonais/imunologia , Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/diagnóstico , Encefalite Japonesa/virologia , Testes de Neutralização/métodos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Proteínas do Envelope Viral/química , Vacinas Virais/imunologia
17.
Virol Sin ; 34(2): 168-174, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31020573

RESUMO

As we know more about Zika virus (ZIKV), as well as its linkage to birth defects (microcephaly) and autoimmune neurological syndromes, we realize the importance of developing an efficient vaccine against it. Zika virus disease has affected many countries and is becoming a major public health concern. To deal with the infection of ZIKV, plenty of experiments have been done on selection of neutralizing antibodies that can target the envelope (E) protein on the surface of the virion. However, the existence of antibody-dependent enhancement (ADE) effect might limit the use of them as therapeutic candidates. In this review, we classify the neutralizing antibodies against ZIKV based on the epitopes and summarize the resolved structural information on antibody/antigen complex from X-ray crystallography and cryo-electron microscopy (cryo-EM), which might be useful for further development of potent neutralizing antibodies and vaccines toward clinical use.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Zika virus/imunologia , Animais , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Camundongos
18.
Vet Microbiol ; 232: 79-83, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31030849

RESUMO

Classical swine fever virus (CSFV) envelope glycoprotein Erns has been shown to bind to cell surface sulphated-heparin-like glycosaminoglycans (GAGs), which participate in cell attachment of the virus. In this study, the CSFV Erns gene was codon optimized for expression in the yeast Pichia pastoris. A C-terminally truncated Erns recombinant protein lacking the previously identified heparin-binding domain (HBD) bound to heparin column, suggesting the presence of another HBD in CSFV Erns. Sequence analyses of the CSFV Erns coding region revealed a common potential N-terminal HBD at residues 301-311. Site-directed mutagenesis of the basic amino acids at K303 and K306 significantly reduced the heparin-binding affinity of the protein. Further mutations of both T310 and H311 had little effect. Thus, a novel potential heparin-binding site near the N-terminus of CSFV strain TD96 Erns has been detected, and the two basic amino acids K303 and K306 are crucial for binding activity to heparin matrix and cell-surface GAGs.


Assuntos
Vírus da Febre Suína Clássica/química , Glicoproteínas/química , Heparina/metabolismo , Proteínas do Envelope Viral/química , Aminoácidos/metabolismo , Sítios de Ligação , Vírus da Febre Suína Clássica/genética , Glicoproteínas/genética , Mutagênese Sítio-Dirigida , Mutação , Fases de Leitura Aberta , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas do Envelope Viral/genética
19.
Nat Microbiol ; 4(7): 1231-1241, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30936489

RESUMO

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Vale do Rift/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Cristalografia por Raios X , Epitopos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Domínios Proteicos , Febre do Vale de Rift/imunologia , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Ligação Viral
20.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
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