Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein
Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein
复制标题
从克隆的互补 DNA 中表达塞姆利基森林病毒蛋白。
作者：
C. Kondor;B. Burke;H. Garoff
将编码Semliki森林病毒（SFV）结构蛋白的互补（cDNA）分子插入到猴病毒40衍生的缺乏内含子的真核表达载体中。将重组DNA导入幼仓鼠肾细胞的细胞核中导致合成真实的SFV膜糖蛋白E1和E2。在用低pH培养基短暂处理细胞后，糖蛋白被转运到细胞表面并诱导细胞-细胞融合。融合反应的pH依赖性与病毒颗粒诱导的相同（白色，J.，J. Kartenbeck和A. Helenius，1980，细胞生物学杂志，89：674-679）。用另一种重组DNA分子转染细胞，其中SFV cDNA被工程化到包括内含子的相同表达载体中，之前已经显示导致仅E2蛋白在细胞表面上表达，而E1蛋白被困在粗面内质网中（Kondor-Koch，C.，H.里德尔，K。Soderberg和H. Garoff，1982，Proc. Natl. Acad. Sci. USA，79：4525-4529）。这些细胞不表现出pH依赖性的多核体形成，这表明E1蛋白是融合活性所必需的。免疫印迹实验表明，从具有内含子的DNA构建体表达的RER捕获的E1蛋白具有比真实E1更小的表观分子量，并且已经失去了其两亲性特征。
A complementary (cDNA) molecule encoding the structural proteins of Semliki Forest virus (SFV) has been inserted into a Simian virus 40- derived eucaryotic expression vector lacking introns. Introduction of the recombinant DNA into nuclei of baby hamster kidney cells results in the synthesis of authentic SFV membrane glycoproteins E1 and E2. The glycoproteins are both transported to the cell surface and induce cell- cell fusion after a brief treatment of the cells with low pH medium. The pH dependence of the fusion reaction was the same as that induced by virus particles (White, J., J. Kartenbeck, and A. Helenius, 1980, J. Cell Biol., 89:674-679). Transfection of cells with another recombinant DNA molecule in which the SFV cDNA is engineered into the same expression vector including an intron has been shown before to result in the expression of only the E2 protein on the cell surface, whereas the E1 protein is trapped in the rough endoplasmic reticulum (Kondor- Koch, C., H. Riedel, K. Soderberg, and H. Garoff, 1982, Proc. Natl. Acad. Sci. USA, 79:4525-4529). Such cells do not exhibit pH-dependent polykaryon formation, suggesting that the E1 protein is necessary for fusion activity. Immunoblotting experiments show that the RER-trapped E1 protein expressed from the DNA construction with an intron has a smaller apparent molecular weight than authentic E1, and that is has lost its amphipathic characteristics.
DOI：
10.1073/pnas.79.4.968
发表时间：
1982-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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作者：
SKEHEL, JJ;BAYLEY, PM;WILEY, DC
通讯作者：
WILEY, DC