RESUMEN
Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design.
Asunto(s)
Epítopos/química , Hepacivirus/inmunología , Hepatitis C/inmunología , Vacunas Sintéticas/química , Proteínas del Envoltorio Viral/síntesis química , Anticuerpos Antivirales/inmunología , Diseño de Fármacos , Epítopos/genética , Epítopos/inmunología , Hepacivirus/química , Hepacivirus/genética , Hepatitis C/prevención & control , Hepatitis C/virología , Humanos , Imitación Molecular , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/química , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
The transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far. Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 - encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1-35) and the transmembrane-intracellular fragment (residues 36-75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer. Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structure-based insight in its mechanism of action which might be helpful in designing new drugs against viral infections.
Asunto(s)
Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas del Envoltorio Viral/análisis , Proteínas Virales/análisis , Animales , Bovinos , Conformación Proteica , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/aislamiento & purificación , Proteínas Virales/síntesis química , Proteínas Virales/aislamiento & purificaciónRESUMEN
Equine infectious anemia (EIA) is a disease caused by a Lentivirus that is currently controlled exclusively by identification of seropositive animals. In most countries, including Brazil, the official diagnostic test for EIA is the agar gel immunodiffusion test (AGID). Although this assay has a high specificity it can produce false negative reactions or equivocal results due to weak precipitation lines, especially in samples from donkeys, mules or newly infected equids. In this pioneering study, it was used overlapping synthetic peptide pools to map and identify a consensus, widely recognised antibody epitope within env encoding the EIAV envelope proteins. A 20-mer soluble peptide encompassing this epitope (pgp45) was then synthesized and tested in an indirect ELISA test. Using a panel of 859 EIA positive and negative equid serum samples, the pgp45 ELISA had 96.1% concordance, 98.6% sensitivity and 95.6% specificity respectively, when compared to AGID. The sensitivity and specificity of the pgp45 ELISA was also >90% when tested in individual equid species including horses (Equus caballus), donkeys (Equus asinus) and mules (Equus caballus x Equus asinus). Moreover, in a horse experimentally infected with the pathogenic Wyoming EIAV strain viral-specific antibodies were detected at 10 days post-infection (dpi) whereas in AGID no specific antibody was detected until 18 days of experimental infection. This peptide can now be used as an antigen in serological tests, especially for rapid screening of large numbers of equids, where it may contribute significantly in the control of EIA, especially at sites with high populations of donkeys and mules.
Asunto(s)
Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Equidae/virología , Anemia Infecciosa Equina/diagnóstico , Caballos/virología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/química , Equidae/inmunología , Anemia Infecciosa Equina/inmunología , Reacciones Falso Negativas , Caballos/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas/veterinaria , Proteínas del Envoltorio Viral/síntesis químicaRESUMEN
Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor α (PILRα) on immune cells. PILRα belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)-like family, members of which bind SA. PILRα is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILRα complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILRα binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILRα. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILRα complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILRα and for the rational design of herpes simplex virus-1 entry inhibitors.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Sitios de Unión , Calorimetría , Cristalografía por Rayos X , Escherichia coli/metabolismo , Humanos , Cinética , Ligandos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Polisacáridos/síntesis química , Polisacáridos/química , Polisacáridos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Proteínas Recombinantes , Estereoisomerismo , Termodinámica , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/químicaRESUMEN
To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.
Asunto(s)
Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Herpesvirus Équido 4/aislamiento & purificación , Enfermedades de los Caballos/virología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Técnicas de Química Sintética , Ensayo de Inmunoadsorción Enzimática/métodos , Caballos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Sensibilidad y Especificidad , Proteínas del Envoltorio Viral/síntesis químicaRESUMEN
Cerenkov luminescence imaging (CLI) is an emerging preclinical molecular imaging modality that tracks the radiation emitted in the visible spectrum by fast moving charged decay products of radionuclides. The aim of this study was in vitro and in vivo evaluation of the two radiotracers, (90) Y-DOTA-PEG28 -A20FMDV2 ((90) Y-1) and (90) Y-DOTA-Ahx-A20FMDV2 ((90) Y-2) (>99% radiochemical purity, 3.7 GBq/µmol specific activity) for noninvasive assessment of tumors expressing the integrin αv ß6 and their future use in tumor targeted radiotherapy. Cell binding and internalization in αv ß6 -positive cells was (90) Y-1: 10.1 ± 0.8%, 50.3 ± 2.1%; (90) Y-2: 22.4 ± 1.7%, 44.7 ± 1.5% with <5% binding to αv ß6 -negative control cells. Biodistribution studies showed maximum αv ß6 -positive tumor uptake of the radiotracers at 1-h post injection (p.i.) ((90) Y-1: 0.64 ± 0.15% ID/g; (90) Y-2: 0.34 ± 0.11% ID/g) with high renal uptake (>25% ID/g at 24 h). Because of the lower tumor uptake and high radioactivity accumulation in kidneys (that could not be reduced by pre-administration of either lysine or furosemide), the luminescence signal from the αv ß6 -positive tumor was not clearly detectable in CLI images. The studies suggest that CLI is useful for indicating major organ uptake for both radiotracers; however, it reaches its limitation when there is low signal-to-noise ratio.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Luminiscencia , Neoplasias/diagnóstico por imagen , Fragmentos de Péptidos/farmacocinética , Radiofármacos/farmacocinética , Radioisótopos de Itrio/farmacocinética , Animales , Integrinas/antagonistas & inhibidores , Masculino , Ratones , Neoplasias/metabolismo , Fragmentos de Péptidos/síntesis química , Unión Proteica , Radiografía , Radiofármacos/síntesis química , Distribución Tisular , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/fisiología , Radioisótopos de Itrio/químicaRESUMEN
Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.
Asunto(s)
Antivirales/administración & dosificación , Virus del Dengue/efectos de los fármacos , Dengue/genética , Péptidos/administración & dosificación , Antivirales/síntesis química , Dengue/tratamiento farmacológico , Dengue/virología , Virus del Dengue/patogenicidad , Humanos , Péptidos/síntesis química , Péptidos/genética , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/genética , Internalización del Virus/efectos de los fármacosRESUMEN
The GB virus C/hepatitis G virus (GBV C/HGV) is a Flaviviridae member that despite its non pathogenicity, has become of great interest given that it could inhibit the replication of the human immunodeficiency virus (HIV). Therefore, a better knowledge of the virus peptides involved in the cellular membrane fusion mechanism has become our aim. The selected peptide, named E2(347-363), corresponds to the GBV-C/HGV E2 protein and has been synthesized in order to study its interaction with in vitro membrane models. Two phospholipids, varying the charge and the unsaturations of the hydrocarbon chain have been chosen: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG). For our porpoise, we have used the Langmuir monolayer technique and Brewster angle microscopy (BAM) to gain deeper insight into the peptide/lipid interactions. The results obtained allow us to argue in favour of considering E2(347-363) a success candidate for developing further experiments in order to determine its potential role in the GBV C/HGV virus/cell membrane fusion process.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Membranas Artificiales , Fragmentos de Péptidos/metabolismo , Fosfatidilgliceroles/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Virus GB-C , Modelos Moleculares , Presión , Propiedades de Superficie , Proteínas del Envoltorio Viral/síntesis químicaRESUMEN
The physicochemical characterization of the peptide sequence E1(145-162) corresponding to the structural protein E1 of the hepatitis G virus was done by studying its interaction with model membranes. Small unilamellar vesicles (SUVs) of dimyristoylphosphatidylglycerol or dimyristoylphosphatidylcholine were chosen as mimetic membranes. Peptide incorporation and location in the phospholipid bilayer was investigated by fluorescence anisotropy with SUVs labeled with diphenylhexatriene (DPH) or trimethylammonium-DPH. The addition of the peptide E1(145-162) showed significant changes in the anisotropy values of the probe located at the air/water interface. These results indicate that the peptide E1(145-162) preferably interacts with the lipid surface without penetrating inside the bilayer. A series of fluorescence experiments based on tryptophan peptide fluorescence were modeled by means of multivariate curve resolution-alternating least squares (MCR-ALS) algorithm to further study the peptide interaction with bilayers at different temperatures. The preliminary results obtained with MCR-ALS showed how the peptide concentration decay is directly linked to the appearance of a new specie, which corresponds to the lipid-peptide binding. These results provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host.
Asunto(s)
Fluorescencia , Liposomas Unilamelares/química , Proteínas del Envoltorio Viral/química , Algoritmos , Dimiristoilfosfatidilcolina/química , Difenilhexatrieno/química , Análisis de los Mínimos Cuadrados , Membrana Dobles de Lípidos/química , Análisis Multivariante , Fosfatidilgliceroles/química , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Propiedades de Superficie , Temperatura , Compuestos de Trimetilamonio/química , Proteínas del Envoltorio Viral/síntesis químicaRESUMEN
Chimeric peptides are unnatural constructs consisting of bioactive compounds from at least two different peptide(s) and/or protein(s) or two sequences from different parts of the same protein. Such multifunctional peptide combinations are prepared to enhance the biological activity or selectivity of their components. New biological effects can also be achieved with the chimera. In this chapter the synthesis of three different types of chimeric peptides will be described. In a linear chimera, two peptide epitopes from different parts of glycoprotein D (gD) of herpes simplex virus (HSV) are combined. A branched chimera, built from linear peptides, consists of tuftsin oligomers with immunostimulatory activity and an epitope peptide of HSV gD. The third compound is a cyclic chimeric molecule, where alpha-conotoxin GI as a host peptide is modified by the incorporation of a core epitope from HSV gD as a guest sequence.
Asunto(s)
Biología Molecular/métodos , Proteínas Recombinantes de Fusión/síntesis química , Secuencia de Aminoácidos , Animales , Conotoxinas/síntesis química , Conotoxinas/química , Conotoxinas/genética , Herpesvirus Humano 1/química , Herpesvirus Humano 1/genética , Humanos , Datos de Secuencia Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Tuftsina/síntesis química , Tuftsina/química , Tuftsina/genética , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 microM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of <25 microM) and cross-inhibition of WNV fusion/infectivity (>99% inhibition at <25 microM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection.
Asunto(s)
Antígenos Virales/farmacología , Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Péptidos/farmacología , Proteínas del Envoltorio Viral/farmacología , Virus del Nilo Occidental/efectos de los fármacos , Secuencia de Aminoácidos , Antígenos Virales/química , Antivirales/síntesis química , Antivirales/química , Línea Celular , Humanos , Modelos Químicos , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Estructura Terciaria de Proteína/genética , Alineación de Secuencia , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/química , Proteínas Virales de Fusión , Acoplamiento Viral/efectos de los fármacosRESUMEN
This paper describes mapping of antigenic and host-protective epitopes of infectious bronchitis virus proteins by assessing the ability of defined peptide regions within the S1, S2 and N proteins to elicit humoral, cell-mediated and protective immune responses. Peptides corresponding to six regions in the S1 (Sp1-Sp6), one in the S2 (Sp7) and four in the N protein (Np1-Np4) were synthesized and coupled to either diphtheria toxoid (dt) or biotin (bt). Bt-peptides were used to assess if selected regions were antigenic and contained B- or T-cell epitopes and dt-peptides if regions induced an antibody response and protection against virulent challenge. All S1 and S2 peptides were antigenic, being recognised by IBV immune sera and also induced an antibody response following inoculation into chicks. Three S1-and one S2-bt peptides also induced a delayed type hypersensitivity response indicating the presence of T-cell epitopes. The S2 peptide Sp7 (amino acid position 566-584) previously identified as an immundominant region, was the most antigenic of all peptides used in this study. Two S1 (Sp4 and Sp6) and one S2 peptide (Sp7), protected kidney tissue against virulent challenge. From four N peptides located in the amino-terminal part of the N protein, only one, Np2 (amino acid position 72-86), was antigenic and also induced a delayed type hypersensitivity response. None of the N peptides induced protection against virulent challenge. The results suggest that the S1 glycoprotein carries additional antigenic regions to those previously identified and that two regions located in the S1 and one in the S2 at amino acid positions 294-316 (Sp4), 532-537 (Sp6) and 566-584 (Sp7) may have a role in protection.
Asunto(s)
Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Epítopos/aislamiento & purificación , Virus de la Bronquitis Infecciosa/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Nucleocápside/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , Pollos , Proteínas de la Nucleocápside de Coronavirus , Epítopos/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/aislamiento & purificación , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/aislamiento & purificación , Hipersensibilidad Tardía , Inmunización , Epítopos Inmunodominantes/inmunología , Masculino , Glicoproteínas de Membrana/síntesis química , Glicoproteínas de Membrana/química , Proteínas de la Nucleocápside/síntesis química , Proteínas de la Nucleocápside/química , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/químicaRESUMEN
We have identified a synthetic peptide derived from the secreted portion of HSV type 2 glycoprotein G, denoted gG-2p20, which has proinflammatory properties in vitro. The gG-2p20 peptide, corresponding to aa 190-205 of glycoprotein G-2, was a chemoattractant for both monocytes and neutrophils in a dose-dependent fashion, and also induced the release of reactive oxygen from these cells. The receptor mediating the responses was identified as the formyl peptide receptor. The gG-2p20-induced activation of phagocytes had a profound impact on NK cell functions. The reactive oxygen species produced by gG-2p20-activated phagocytes both inhibited NK cell cytotoxicity and accelerated the apoptotic cell death in NK cell-enriched lymphocyte populations. Hence, we have for the first time been able to identify a potential function of the secreted portion of HSV-2 glycoprotein G. We propose that the proinflammatory gG-2p20 peptide identified could contribute to a reduced function and viability of NK cells during HSV-2 infection due to its ability to recruit and activate phagocytic cells.
Asunto(s)
Herpesvirus Humano 2/inmunología , Mediadores de Inflamación/fisiología , Células Asesinas Naturales/inmunología , Monocitos/inmunología , Neutrófilos/inmunología , Fragmentos de Péptidos/fisiología , Proteínas del Envoltorio Viral/fisiología , Secuencia de Aminoácidos , Apoptosis/inmunología , Quimiotaxis de Leucocito/inmunología , Citotoxicidad Inmunológica/inmunología , Inducción Enzimática/inmunología , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/inmunología , Mediadores de Inflamación/síntesis química , Células Asesinas Naturales/patología , Células Asesinas Naturales/virología , Activación de Macrófagos/inmunología , Datos de Secuencia Molecular , Monocitos/patología , Monocitos/virología , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/metabolismo , Activación Neutrófila/inmunología , Neutrófilos/enzimología , Neutrófilos/patología , Neutrófilos/virología , Fragmentos de Péptidos/síntesis química , Toxina del Pertussis/farmacología , Fagocitos/enzimología , Fagocitos/inmunología , Fagocitos/patología , Fagocitos/virología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Formil Péptido/fisiología , Proteínas del Envoltorio Viral/síntesis químicaRESUMEN
Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emergent virus responsible for a worldwide epidemic in 2003. The coronavirus spike proteins belong to class I fusion proteins, and are characterized by the existence of two heptad repeat (HR) regions, HR1 and HR2. The HR1 region in coronaviruses is predicted to be considerably longer than that in other type I virus fusion proteins. Therefore the exact binding sequence to HR2 from the HR1 is not clear. In this study, we defined the region of HR1 that binds to HR2 by a series of biochemical and biophysical measures. Subsequently the defined HR1 (902-952) and HR2 (1145-1184) chains, which are different from previously defined binding regions, were linked together by a flexible linker to form a single-chain construct, 2-Helix. This protein was expressed in Escherichia coli and forms a typical six-helix coiled coil bundle. Highly conserved HR regions between mouse hepatitis virus (MHV) and SARS-CoV spike proteins suggest a similar three-dimensional structure for the two fusion cores. Here, we constructed a homology model for SARS coronavirus fusion core based on our biochemical analysis and determined the MHV fusion core structure. We also propose an important target site for fusion inhibitor design and several strategies, which have been successfully used in fusion inhibitor design for human immunodeficiency virus (HIV), for the treatment of SARS infection.
Asunto(s)
Glicoproteínas de Membrana/síntesis química , Modelos Moleculares , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/síntesis química , Secuencias Repetitivas de Aminoácido , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Proteínas del Envoltorio Viral/síntesis química , Proteínas Virales de Fusión/síntesis química , Secuencia de Aminoácidos , Sitios de Unión/genética , Simulación por Computador , Vectores Genéticos/síntesis química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Virus de la Hepatitis Murina/química , Virus de la Hepatitis Murina/genética , Virus de la Hepatitis Murina/patogenicidad , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Unión Proteica/genética , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Aminoácido/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Solubilidad , Glicoproteína de la Espiga del Coronavirus , Homología Estructural de Proteína , Proteínas del Envoltorio Viral/genética , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is not only responsible for receptor binding and virus fusion, but also a major Ag among the SARS-CoV proteins that induces protective Ab responses. In this study, we showed that the S protein of SARS-CoV is highly immunogenic during infection and immunizations, and contains five linear immunodominant sites (sites I to V) as determined by Pepscan analysis with a set of synthetic peptides overlapping the entire S protein sequence against the convalescent sera from SARS patients and antisera from small animals immunized with inactivated SARS-CoV. Site IV located in the middle region of the S protein (residues 528-635) is a major immunodominant epitope. The synthetic peptide S(603-634), which overlaps the site IV sequence reacted with all the convalescent sera from 42 SARS patient, but none of the 30 serum samples from healthy blood donors, suggesting its potential application as an Ag for developing SARS diagnostics. This study also provides information useful for designing SARS vaccines and understanding the SARS pathogenesis.
Asunto(s)
Mapeo Epitopo/métodos , Epítopos Inmunodominantes/análisis , Glicoproteínas de Membrana/inmunología , Síndrome Respiratorio Agudo Grave/diagnóstico , Síndrome Respiratorio Agudo Grave/prevención & control , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Biología Computacional/métodos , Humanos , Epítopos Inmunodominantes/administración & dosificación , Epítopos Inmunodominantes/inmunología , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/síntesis química , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Estructura Terciaria de Proteína/genética , Conejos , Juego de Reactivos para Diagnóstico , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Síndrome Respiratorio Agudo Grave/inmunología , Glicoproteína de la Espiga del Coronavirus , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/síntesis química , Vacunas de Productos Inactivados/inmunología , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/síntesis química , Vacunas Virales/administración & dosificación , Vacunas Virales/síntesis químicaRESUMEN
A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S(1190)). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S(1190) binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S(1190) maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S(1190) glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins.
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Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Aminoácidos/química , Aminoácidos/genética , Animales , Línea Celular , Chlorocebus aethiops , Codón , Citometría de Flujo , Humanos , Ligandos , Glicoproteínas de Membrana/síntesis química , Glicoproteínas de Membrana/genética , Mutación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Glicoproteína de la Espiga del Coronavirus , Relación Estructura-Actividad , Células Vero , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/genéticaRESUMEN
The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells. It is a type 1 viral fusion protein that characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein. Previous studies have shown that the two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers), which mediates fusion of viral and host cell membranes. Here we describe the biophysical analysis of the two predicted heptad repeat regions within the severe acute respiratory syndrome coronavirus S protein. Our results show that in isolation the HR-N region forms a stable alpha-helical coiled coil that associates in a tetrameric state. The HR-C region in isolation formed a weakly stable trimeric coiled coil. When mixed together, the two peptide regions (HR-N and HR-C) associated to form a very stable alpha-helical 6-stranded structure (trimer of heterodimers). Systematic peptide mapping showed that the site of interaction between the HR-N and HR-C regions is between residues 916-950 of HR-N and residues 1151-1185 of HR-C. Additionally, interchain disulfide bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel. Overall, the structure of the hetero-stranded complex is consistent with the structures observed for other type 1 viral fusion proteins in their fusion-competent state.
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Glicoproteínas de Membrana/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , Cartilla de ADN , Escherichia coli/genética , Hemaglutininas Virales/química , Hemaglutininas Virales/aislamiento & purificación , Glicoproteínas de Membrana/síntesis química , Glicoproteínas de Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Conformación Proteica , Mapeo Restrictivo , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/síntesis química , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
The results of conformational analysis of linear and cyclic peptides from the 276SALLEDPVG(284) sequence of glycoprotein D of Herpes simplex virus are presented. The epitope peptides were synthesized by SPPS and on resin cyclization was applied for preparation of cyclic compounds. Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and nuclear magnetic resonance (NMR) were used to determine of the solution structure of both linear and cyclic peptides. The results indicated that the cyclopeptides containing the core of the epitope (DPVG) as a part of the cycle have more stable beta-turn structure than the linear peptides or the cyclic analogues, where the core motif is not a part of the cycle. NMR study of H-SALLc(EDPVGK)-NH(2) confirm presence of a type I beta-turn structure which includes the DPVG epitope core.
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Epítopos , Péptidos Cíclicos/química , Proteínas del Envoltorio Viral/química , Dicroismo Circular , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/síntesis química , Estructura Secundaria de Proteína , Análisis de Secuencia de Proteína , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectroscopía Infrarroja por Transformada de Fourier , Proteínas del Envoltorio Viral/síntesis químicaRESUMEN
Six peptide fragments of the envelope protein E of the tick-borne encephalitis virus involving the predicted T-helper epitopes were synthesized. Their ability to induce antibodies without conjugation with any high-molecular-mass carrier was studied in mice of three lines. Five of six synthesized peptides exhibited immunogenic properties, which differed in dependence on the haplotype of immunized mice. The peptide binding to the antiviral antibodies was studied, and two peptides were revealed that demonstrated a high ability to recognize the viral antibodies in the horse and human sera. These peptides are promising for the development of diagnostic agents for the tick-borne encephalitis virus.
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Proteínas del Envoltorio Viral/síntesis química , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/química , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/aislamiento & purificación , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276-284 region of HSV gD-1 selected for these studies is highly hydrophilic and adopts a beta-turn. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, stabilized by two disulfide bridges at positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, 8Arg-His-Tyr-Ser12 has been replaced by Asp-Pro-Val-Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3-13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys2-Cys7 three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 M HCl or Tl(tfa)3 in trifluoroacetic acid (TFE)] were compared. The high-performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV-alpha-[Tyr1]-conotoxin chimeric peptide and native alpha-conotoxin GI showed similar circular dichroism spectra in phosphate-buffered saline (PBS) and in a PBS-TFE 1:1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M- and IgG-type antibody responses showed that the bicyclic HSV-alpha-[Tyr1]-conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV-alpha-[Tyr1]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV-alpha-[Tyr1]-conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG-specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.