Peränen J, Laakkonen P, Hyvönen M, Kääriäinen L
Institute of Biotechnology, University of Helsinki, Finland.
Virology. 1995 Apr 20;208(2):610-20. doi: 10.1006/viro.1995.1192.
In alphavirus-infected cells the four virus-specific nonstructural proteins (nsP1-nsP4) are located on modified endosomes and lysosomes known as type I cytopathic vacuoles. In this paper, we show that when nsP1 was expressed alone in HeLa cells with the aid of the recombinant T7 RNA polymerase vaccinia (vTF7-3) virus system, it was tightly associated with intracellular smooth membranes. The membrane association may be due to acylation, since nsP1 could be labeled with [3H]palmitic acid in both Semliki Forest virus-infected and nsP1-transfected HeLa cells. Release of the 3H-label by alkaline methanolysis suggests that the palmitate was associated with nsP1 via an ester bond. Pulse-chase experiments done on nsP1-transfected cells revealed that this protein was rapidly associated with the membranes. After synchronizing the synthesis of the nsP1 gene product in transfected cells, nsP1 appeared first at the plasma membrane and thereafter on vesicles, many of which contained the endosomal transferrin receptor marker. Later, nsP1 appeared on large vacuoles, which contained the lysosome specific h-lamp-1 protein. Membrane association of nsP1, and its affinity to endosomes and lysosomes, suggest a role of this protein in the biogenesis of the alphavirus-specific RNA replication complex.
在甲病毒感染的细胞中，四种病毒特异性非结构蛋白（nsP1 - nsP4）定位于被称为I型细胞病变空泡的修饰内体和溶酶体上。在本文中，我们表明，当借助重组T7 RNA聚合酶痘苗病毒（vTF7 - 3）系统在HeLa细胞中单独表达nsP1时，它与细胞内的光滑膜紧密相关。这种膜结合可能是由于酰化作用，因为在塞姆利基森林病毒感染的细胞和nsP1转染的HeLa细胞中，nsP1都可以用[3H]棕榈酸标记。碱性甲醇解使3H标记释放，这表明棕榈酸通过酯键与nsP1相连。对nsP1转染细胞进行的脉冲追踪实验表明，该蛋白迅速与膜结合。在转染细胞中同步nsP1基因产物的合成后，nsP1首先出现在质膜上，随后出现在小泡上，其中许多小泡含有内体转铁蛋白受体标记物。后来，nsP1出现在大空泡上，这些大空泡含有溶酶体特异性的h - lamp - 1蛋白。nsP1的膜结合及其对内体和溶酶体的亲和力表明该蛋白在甲病毒特异性RNA复制复合物的生物发生中起作用。
