RESUMEN
Sindbis virus nucleocapsids were isolated from intact virus particles and from the cytoplasms of infected cells. These two isolates of purified nucleocapsids differed from one another in ribonuclease sensitivity and in sedimentation velocity. These findings suggest that a conformational change takes place in the Sindbis virus nucleocapsid during envelopment.
Asunto(s)
Cápside/análisis , Virus Sindbis/análisis , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Conformación de Ácido Nucleico , Ribonucleasas/farmacologíaRESUMEN
Sindbis virus nucleocapsids were isolated from mature virions by a two-step purification method. Detergent-treated virions were sedimented in sucrose gradients and the nucleocapsid peaks chromatographed on RNase-free Sephadex G-200. The purified nucleocapsids displayed several morphologies when examined in the electron microscope. These morphologies, and the results of double-angle shadowing, suggest that the core of this enveloped virus has the shape of a regular icosahedron with a triangulation number of 4. Peptide mapping of capsid protein obtained from nucleocapsids that had been radioiodinated by a variety of means, indicated that of the four tyrosine residues in the protein, only Tyr180 was exposed at the surface of the icosahedral structure. The other three residues were not exposed on the outer surface of the nucleocapsid shell, nor on the surface of capsid protein itself, implying that they were buried within the folded protein.
Asunto(s)
Cápside/análisis , Virus Sindbis/análisis , Proteínas del Núcleo Viral/análisis , Cápside/aislamiento & purificación , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Microscopía Electrónica , Mapeo Peptídico , Virus Sindbis/genética , Virus Sindbis/ultraestructura , Proteínas del Núcleo Viral/aislamiento & purificaciónRESUMEN
Glycopeptides and endogly cosidase-digested oligosaccharides from [3H]mannose-labeled Rous sarcoma virus and Sinbis virus have been fractionated by lentil lectin-Sepharose and concanavalin A-agarose affinity chromatography and subsequently analyzed by BioGel P-4 gel filtration. Only a specific subset of the Con A-bound asparaginly-oligosaccharides from he two viruses was also bound to lentil lectin, and this freaction apparently represented fucose-containing, diantennary acidic-type structures ((NeuNAc +/- Gal-GlcNAc)2 Man3 -GlcNAc2 (fucose)-ASN). The largest glycopeptides from Rous sarcoma virus were unbound to either Con A or lentil lectin and presumably contained tri- and/or tetra-antennary acidic-type structures ((NeuNAc +/- Gal-GlcNAc)3--4 -Man 3GlcNAc2 (+/- fucose)-ASN). In contrast, the majority of 'hybrid'-type oligosaccharides and essentially all of the neutral oligomannosyl core structures (Man5--9 GlcNAc1 and Man3 GlcNAc1) from the endoglycosidase-digested glycopeptides of both viruses were specifically bound to Con A-agarose, with the largest neutral oligosaccharides (Man7--9GlcNAc1) bound more tightly and less efficiently eluted by alpha-methyl mannoside.
Asunto(s)
Virus del Sarcoma Aviar/análisis , Glicopéptidos/aislamiento & purificación , Oligosacáridos/aislamiento & purificación , Virus Sindbis/análisis , Proteínas Virales/aislamiento & purificación , Fenómenos Químicos , Química , Cromatografía de Afinidad , Cromatografía en Gel , Lectinas , Sefarosa/análogos & derivadosRESUMEN
RNA of partially purified Kemerovo virus sedimented in a relatively broad region in sucrose density gradient with a peak at 14 S. Approximately 65% of this RNA was resistant to the action of ribonuclease which indicates the double-stranded nature of Kemerovo virus RNA.
Asunto(s)
ARN Viral/análisis , Reoviridae/análisis , Animales , Encéfalo/microbiología , Centrifugación por Gradiente de Densidad , Embrión de Pollo , Técnicas de Cultivo , Ratones , Desnaturalización de Ácido Nucleico , ARN Viral/metabolismo , Ratas , Ribonucleasas/metabolismo , Virus Sindbis/análisis , Tritio , Uridina , Cultivo de VirusRESUMEN
In pulse-chase experiments with Karelian fever virus-infected cells, proteins were found with molecular weights of 130, 98, 78, and 62 kD of which the first, second and fourth were classified as polypeptide precursors of the structural proteins of virion. The molecular weights of proteins E1, E2 and C of 52, 47 and 34 kD, respectively, as well as isoelectric points of isolated glycoproteins (pI E1 = 6.3, pI E2 = 8.4) were similar in KFV (strain Leiv-9298) and Sindbis virus (strain AR339). The antigenic similarity of the strains under study in neutralization test with hyperimmune sera, the identity of physicochemical characteristics of the structural proteins of KFV and prototype Sindbis virus strain suggest a close relationship of the Leiv-9298 strain to the Afro-European variants of Sindbis virus.
Asunto(s)
Alphavirus/análisis , Péptidos/análisis , Virus Sindbis/análisis , Alphavirus/inmunología , Alphavirus/aislamiento & purificación , Antígenos Virales/análisis , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida/métodos , Focalización Isoeléctrica/métodos , Peso Molecular , Pruebas de Neutralización , Virus Sindbis/inmunología , Virus Sindbis/aislamiento & purificaciónAsunto(s)
Glicoproteínas , Virus Sindbis/análisis , Proteínas Virales , Virión/análisis , Anticuerpos Antivirales , Especificidad de Anticuerpos , Reactivos de Enlaces Cruzados , Dimetil Suberimidato , Electroforesis en Gel de Poliacrilamida , Precursores de Proteínas , Virus de los Bosques Semliki/análisis , Virus Sindbis/inmunologíaAsunto(s)
Glicoproteínas , Virus Sindbis/análisis , Proteínas Virales , Virión/análisis , Secuencia de Aminoácidos , Aminoácidos/análisis , Cromatografía Líquida de Alta Presión , Quimotripsina , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/aislamiento & purificación , Palmitatos , Proteínas Virales/aislamiento & purificaciónAsunto(s)
Arbovirus/análisis , Mutación , Animales , Centrifugación Zonal , Embrión de Pollo , Pruebas de Fijación del Complemento , Electroforesis Discontinua , Genética Microbiana , Hemabsorción , Pruebas de Hemaglutinación , Biología Molecular , Morfogénesis , Fenotipo , ARN Viral/análisis , ARN Viral/biosíntesis , Virus Sindbis/análisis , Virus Sindbis/crecimiento & desarrollo , Virus Sindbis/metabolismo , Temperatura , Ultrasonido , Cultivo de VirusAsunto(s)
Centrifugación por Gradiente de Densidad/métodos , Colifagos/ultraestructura , ADN Bacteriano/análisis , ADN Viral/análisis , Clostridium perfringens/análisis , Colifagos/análisis , Virus ADN/análisis , Virus ADN/ultraestructura , Ácido Yotalámico , Matemática , Micrococcus/análisis , Microscopía Electrónica , Mutación , ARN Viral/análisis , Virus Sindbis/análisis , Especificidad de la EspecieAsunto(s)
Virus Sindbis/aislamiento & purificación , Adsorción , Centrifugación , Cromatografía , Quimotripsina , Desoxirribonucleasas/metabolismo , Ácidos Nucleicos/análisis , Virus Sindbis/análisis , Virus Sindbis/enzimología , Virus Sindbis/patogenicidad , Tripsina/metabolismo , Ultrafiltración , Ultrasonido , Proteínas Virales/análisis , VirulenciaAsunto(s)
Virus de la Encefalitis Equina del Oeste/análisis , Virus de la Encefalitis/análisis , Encefalomielitis Equina/microbiología , Virus Sindbis/análisis , Proteínas Virales/análisis , Antígenos Virales/análisis , Radioisótopos de Carbono , Colina , Pruebas de Fijación del Complemento , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Virus de la Encefalitis Equina del Oeste/inmunología , Virus de la Encefalitis/inmunología , Glucosamina , Pruebas de Inhibición de Hemaglutinación , Pruebas de Hemaglutinación , Inmunodifusión , Leucina , Lipoproteínas/análisis , Nucleoproteínas/análisis , Virus Sindbis/inmunología , Tensoactivos/farmacología , TritioAsunto(s)
Arbovirus/análisis , Nucleoproteínas/aislamiento & purificación , Proteínas Virales/aislamiento & purificación , Animales , Ácidos y Sales Biliares , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Cesio , Embrión de Pollo , Chlorella , Cloruros , Técnicas de Cultivo , Detergentes , Hemaglutininas Virales/aislamiento & purificación , Hidrolisados de Proteína , Virus Sindbis/análisis , Espectrofotometría , Sacarosa , Tritio , Uridina , Cultivo de VirusAsunto(s)
Arbovirus/análisis , Centrifugación por Gradiente de Densidad , Animales , Antígenos/análisis , Líquido Ascítico , Ácidos y Sales Biliares/farmacología , Tampones (Química) , Cesio , Embrión de Pollo , Cloruros , Pruebas de Fijación del Complemento , Técnicas de Cultivo , Efecto Citopatogénico Viral , Eritrocitos/inmunología , Éteres de Etila/farmacología , Gansos , Pruebas de Inhibición de Hemaglutinación , Pruebas de Hemaglutinación , Hemaglutininas Virales/análisis , Concentración de Iones de Hidrógeno , Ratones , Virus Sindbis/análisis , Virus Sindbis/efectos de los fármacos , Virus Sindbis/inmunología , Virus Sindbis/patogenicidad , Especificidad de la Especie , Tensoactivos/farmacología , Trometamina , Proteínas Virales/análisisAsunto(s)
Virosis/microbiología , Línea Celular , Densitometría , Genes Virales , Virus Helper , Humanos , Interferones/fisiología , Peso Molecular , Hibridación de Ácido Nucleico , ARN Viral/análisis , ARN Viral/genética , Rubéola (Sarampión Alemán)/microbiología , Virus Sindbis/análisis , Virus Sindbis/genética , Virus Sindbis/crecimiento & desarrollo , Factores de Tiempo , Interferencia Viral , Replicación ViralAsunto(s)
Glicoproteínas/biosíntesis , Proteínas Virales/biosíntesis , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Glicósidos/análisis , Orthomyxoviridae/análisis , Retroviridae/análisis , Virus de los Bosques Semliki/análisis , Virus Sindbis/análisis , Virus de la Estomatitis Vesicular Indiana/análisisRESUMEN
Selective binding of lipid to glycoprotein was detected when [3H]palmitate-labeled Sindbis virus particles or viral-infected cells were disrupted by heating with sodium dodecyl sulfate, and glycoproteins were isolated by electrophoresis in sodium dodecyl sulfate/10% polyacrylamide gels. The smaller glycoprotein (E2) retained 2 to 3 times more labeled lipid than did the larger EI glycoprotein, and the cell-associated glycoprotein precursor (PE2) bound even less lipid. No lipid was associated with the nonglycosylated glycoproteins that accumulated in infected cells treated with tunicamycin. The labeled lipid remained bound to the glycoproteins after exhaustive extraction with chloroform/methanol of virus particles, infected-cell extracts, or isolated glycoproteins, but it could be extracted by chloroform/methanol after treating glycoproteins with mild alkali. Analysis by gas/liquid chromatography showed that 60% of the label was in palmitate and the balance of label was distributed between oleate and stearate. There were approximately 2 mol of fatty acid bound per mol of E1 glycoprotein. Proteolysis of the fatty acid-labeled glycoprotein with pepsin, thermolysin, and Pronase degraded the polypeptide to fragments that retained the fatty acids in an alkali-labile state. These data suggest that a covalent attachment of fatty acid may occur during maturation of the viral glycoproteins.
Asunto(s)
Ácidos Grasos/análisis , Glicoproteínas , Virus Sindbis/análisis , Proteínas Virales , Ácidos Palmíticos , Fragmentos de Péptidos , Pronasa , Unión ProteicaRESUMEN
The lipid-linked oligosaccharide synthesized in vitro, in the presence of 1.0 microM UDP-[3H]Glc, GDP-[14C]Man, and UDP-GlcNAc has been isolated and the structure of the oligosaccharide has been analyzed. The oligosaccharide contains 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues. The N-acetylglucosamine residues are located at the reducing terminus. The 3 glucose residues are arranged in a linear order at one of the nonreducing termini in the sequence Glc 1,2--Glc 1,3--Glc--(Man)9 (GlcNAc)2. The structural analysis was made possible largely by the availability of glucosidase preparations of fungal anad microsomal origin which remove glucose residues from the oligosaccharide without releasing mannose residues.
Asunto(s)
Asparagina , Glucosa/análisis , Oligosacáridos , Línea Celular , Manosidasas , Metilación , Oxidación-Reducción , Ácido Peryódico , Virus Sindbis/análisisRESUMEN
Oligonucleotide mapping studies of the RNA from standard and defective interfering particles of Sindbis virus demonstrate that 3'- and 5'-terminal regions of the genome are conserved in the defective RNAs. These studies also suggest that defective RNAs contain multiple deletions.
Asunto(s)
Virus Defectuosos/análisis , Oligonucleótidos/análisis , ARN Viral/análisis , Virus Sindbis/análisis , Secuencia de BasesRESUMEN
The single late 26S mRNA of Semliki Forest virus (SFV) directs the synthesis of the four viral structural proteins, C, E3, E2, and E1, and the recently described nonstructural protein, 6K. We report here partial NH2-terminal amino acid sequences of the SFV polypeptides E3 and 6K and of p62, the precursor to E3 and E2. In addition, were have determined a partial NH2-terminal sequence of the Sindbis virus homolog of 6K, the 4.2K protein. p62 and E3 of SFV have identical NH2-terminal amino acid sequences. Comparison of the partial NH2-terminal sequences of 6K of SFV and 4.2K of Sindbis virus with the deduced amino acid sequence encoded by the 26S mRNA of each virus reveals that the genes for these peptides are located in each case between those for E2 and E1. The order of the genes on the 26S mRNA of the alphaviruses is therefore 5'-C-E3-E2-6K-E1-3'. We discuss two mechanisms by which the nascent viral glycoproteins may be inserted into the membrane of the endoplasmic reticulum.