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首页> 外文期刊>Journal of Virology >Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells.
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Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells.

机译：鉴定SINDBIS病毒免疫交叉反应性蛋白质：用于从感染细胞的E1糖蛋白独特构象的证据。

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Hyperimmune antisera to purified Sindbis (SIN) or Semliki Forest (SF) virus were used to identify alphavirus-specific and cross-reactive proteins in virions and infected cells. The hyperimmune sera participated in homologous and cross-cytolysis of alphavirus-infected cells, and the use of monospecific antisera to SIN structural proteins suggested that E1 and E2 could serve as target proteins in cytolysis. Proteins from purified virions or infected cells were extracted with Nonidet P-40, denatured by procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose solid supports, and reacted with hyperimmune sera and 125I-labeled protein A (immunoblotting on denatured proteins). Alternatively, native proteins extracted by mild Nonidet P-40 treatment were precipitated with hyperimmune sera before denaturation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After immunoblotting, homologous antiserum reacted with the virus structural proteins E1, E2, capsid extracted from purified virions, and the counterparts of these proteins extracted from infected cells. In addition, PE2 and a 92,000-molecular-weight protein from infected cells reacted with homologous antiserum. These proteins were also immunoprecipitated with homologous antiserum. After immunoblotting, the Sindbis capsid protein was shown to be cross-reactive whether derived from purified virions or from infected cells; no cross-reactivity was observed with PE2 or E2 from either source, and the E1 glycoprotein was shown to be cross-reactive only when obtained from virions. However, the E1 glycoprotein could be cross-immunoprecipitated from infected cells (as well as from disrupted virions), and, in addition, capsid and a 92,000-molecular-weight protein were cross-immunoprecipitated from infected cells. These results suggest that a native conformation of the cell-associated E1 glycoproteins may be required for immunological cross-reactivity (immune precipitation), whereas virion but not cell-associated E1 retains immunological cross-reactivity after denaturation (immunoblot technique). The findings extend our previously published evidence which suggested that alphavirus maturation is accompanied by a change in immunological cross-reactivity with respect to E1.
机译：超导抗血清纯化的SINDBIS（SIN）或Semliki森林（SF）病毒用于鉴定病毒粒子和感染细胞中的alphaVirus特异性和交叉反应性蛋白质。 HyperiMMune血清参与了alphavirous感染细胞的同源和交叉细胞分解，并且使用单特异性抗血清至Sin结构蛋白表明E1和E2可以作为细胞分解中的靶蛋白。用NATIDED P-40萃取来自纯化的病毒病毒病毒藻或感染细胞的蛋白质，通过向硫酸钠 - 聚丙烯酰胺凝胶电泳的程序进行变性，转移到硝酸纤维素固体载体中，并与超微征血清和125i标记的蛋白A反应（变性蛋白质上的免疫印迹）反应。或者，在通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳的变性之前，用温和的NORIDET P-40处理中提取的天然蛋白质用超微静脉血清沉淀。在免疫印迹之后，同源抗血清与从纯化的病毒粒中提取的病毒结构蛋白E1，E2，衣壳中的病毒结构蛋白E1，以及从受感染的细胞中提取的这些蛋白质的对应物。另外，来自感染细胞的PE2和92,000分子量蛋白质与同源抗血清反应。这些蛋白质也用同源抗血清免疫沉淀。在免疫印迹后，证明SINDBIS衣壳蛋白是否衍生自净化的病毒粒子或来自受感染的细胞的交叉反应;没有从任一来源的PE2或E2观察到交叉反应性，并且E1糖蛋白仅当从病毒粒中获得时是交叉反应性的。然而，E1糖蛋白可以从受感染的细胞（以及来自破坏的病毒粒子）交叉免疫沉淀，并且另外，衣壳和92,000分子量蛋白质从受感染的细胞交叉免疫沉淀。这些结果表明，免疫交叉反应性（免疫沉淀）可能需要细胞相关的E1糖蛋白的天然构象，而病毒素而不是细胞相关的E1在变性后保持免疫反应性（免疫斑技术）。调查结果扩展了先前公布的证据，表明alphaVirus成熟伴随着关于E1的免疫交叉反应性的变化。

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