Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells.
Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells.
复制标题
辛德毕斯病毒免疫交叉反应蛋白的鉴定：感染细胞中 E1 糖蛋白独特构象的证据。
DOI：
10.1128/jvi.49.2.379-385.1984
复制
发表时间：
1984
影响因子：
5.4
通讯作者：
Brown,A
中科院分区：
文献类型：
--
作者：
Wolcott,JA;Wust,CJ;Brown,A
使用针对纯化辛德比斯 (SIN) 或塞姆利基森林 (SF) 病毒的超免疫抗血清来鉴定病毒粒子和感染细胞中的甲病毒特异性蛋白和交叉反应蛋白。超免疫血清参与甲病毒感染细胞的同源和交叉细胞溶解，并且使用针对SIN结构蛋白的单特异性抗血清表明E1和E2可以作为细胞溶解中的靶蛋白。用 Nonidet P-40 提取纯化病毒粒子或感染细胞中的蛋白质，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳程序变性，转移到硝酸纤维素固体支持物上，并与超免疫血清和 125I 标记的蛋白 A 反应（对变性蛋白质进行免疫印迹）。或者，通过温和的 Nonidet P-40 处理提取的天然蛋白质用超免疫血清沉淀，然后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳变性。免疫印迹后，同源抗血清与病毒结构蛋白 E1、E2、从纯化病毒粒子中提取的衣壳以及从感染细胞中提取的这些蛋白质的对应物发生反应。此外，PE2 和来自受感染细胞的 92,000 分子量蛋白质与同源抗血清发生反应。这些蛋白质也用同源抗血清进行免疫沉淀。免疫印迹后，辛德比斯衣壳蛋白显示出交叉反应，无论是来自纯化的病毒体还是来自感染的细胞；没有观察到与任一来源的 PE2 或 E2 发生交叉反应，并且 E1 糖蛋白仅在从病毒粒子获得时才显示出交叉反应。然而，E1 糖蛋白可以从受感染的细胞（以及破碎的病毒粒子）中交叉免疫沉淀，此外，衣壳和 92,000 分子量的蛋白质也可以从受感染的细胞中交叉免疫沉淀。这些结果表明，细胞相关 E1 糖蛋白的天然构象可能是免疫交叉反应性（免疫沉淀）所必需的，而病毒粒子而非细胞相关 E1 在变性后保留了免疫交叉反应性（免疫印迹技术）。这些发现扩展了我们之前发表的证据，表明甲病毒的成熟伴随着与 E1 相关的免疫交叉反应性的变化。
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.