Krady J K, Ward D C
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Mol Cell Biol. 1995 Jan;15(1):524-33. doi: 10.1128/MCB.15.1.524.
The nonstructural protein NS-1, encoded by the parvovirus minute virus of mice, is a potent regulator of viral gene expression. NS-1 does not bind DNA in a sequence-specific manner, and the mechanism by which it modulates viral promoter function is unclear. We have used Gal4-NS-1 fusion protein constructs to identify and characterize an activating domain encoded within the C-terminal 88 amino acids of NS-1 which competes effectively with the acidic activator domain of the herpes simplex virus VP16 protein. DNA affinity chromatography and immunoprecipitation experiments demonstrate that protein-protein interactions between the transcription factor Sp1 and NS-1 are required to bind NS-1 to promoter DNA in vitro. Cotransfection of Gal4-NS-1 and Sp1-VP16 acidic activator constructs into Drosophila melanogaster Schneider cells, which lack endogenous Sp1, stimulates transcription from a minimal promoter containing five Gal4 binding sites, while single-construct transfections do not. Cotransfection of Schneider cells with wild-type NS-1 and Sp1 constructs activates transcription from a simian virus 40 promoter 10- to 30-fold over that of either construct alone. Thus, Sp1-NS-1 interactions in vivo can stimulate transcription from a heterologous promoter containing Sp1 binding sites.
由小鼠细小病毒编码的非结构蛋白NS-1是病毒基因表达的有效调节因子。NS-1不以序列特异性方式结合DNA，其调节病毒启动子功能的机制尚不清楚。我们使用Gal4-NS-1融合蛋白构建体来鉴定和表征NS-1 C末端88个氨基酸内编码的一个激活域，该激活域能与单纯疱疹病毒VP16蛋白的酸性激活域有效竞争。DNA亲和层析和免疫沉淀实验表明，转录因子Sp1与NS-1之间的蛋白质-蛋白质相互作用是在体外将NS-1与启动子DNA结合所必需的。将Gal4-NS-1和Sp1-VP16酸性激活构建体共转染到缺乏内源性Sp1的黑腹果蝇Schneider细胞中，可刺激来自含有五个Gal4结合位点的最小启动子的转录，而单构建体转染则不能。将野生型NS-1和Sp1构建体与Schneider细胞共转染，可使猿猴病毒40启动子的转录比单独的任何一种构建体激活10至30倍。因此，体内Sp1-NS-1相互作用可刺激来自含有Sp1结合位点的异源启动子的转录。
