Kawaguchi Y, Tomonaga K, Maeda K, Ono M, Miyazawa T, Kohmoto M, Tohya Y, Mikami T
Department of Veterinary Microbiology, Faculty of Agriculture, University of Tokyo, Japan.
Virology. 1995 Apr 20;208(2):492-9. doi: 10.1006/viro.1995.1180.
We investigated effects of site-specific mutation of the putative C/EBP binding site in the feline immunodeficiency virus (FIV) long terminal repeat (LTR) on the basal promoter activity in Crandell feline kidney (CRFK) cells and on replication efficiency in CRFK cells and a T-lymphoblastoid cell line, MYA-1 cells. Mutation of the C/EBP site reduced the basal promoter activity in CRFK cells and prevented efficient FIV replication in both CRFK and MYA-1 cells. Gel-mobility-shift assay using nuclear extracts from CRFK and MYA-1 cells revealed that the nuclear factor(s) actually binds to the C/EBP site, but there was a clear difference in the binding patterns to the C/EBP site between CRFK and MYA-1 cell nuclear proteins. Furthermore, we demonstrated that the C/EBP site is necessary for inhibition of FIV LTR-directed gene expression by pseudorabies virus (PRV) ICP4. The C/EBP site is sufficient to confer inhibitory effect by PRV ICP4 on heterologous promoters. These data suggest that the C/EBP site in the FIV LTR is important for the positive regulation of FIV gene expression and replication and is also required for the negative regulation of FIV gene expression by PRV ICP4.
我们研究了猫免疫缺陷病毒(FIV)长末端重复序列(LTR)中假定的C/EBP结合位点的位点特异性突变对克兰德尔猫肾(CRFK)细胞中基础启动子活性以及对CRFK细胞和T淋巴母细胞系MYA-1细胞中复制效率的影响。C/EBP位点的突变降低了CRFK细胞中的基础启动子活性,并阻止了FIV在CRFK和MYA-1细胞中的有效复制。使用CRFK和MYA-1细胞核提取物进行的凝胶迁移率变动分析表明,核因子实际上与C/EBP位点结合,但CRFK和MYA-1细胞核蛋白与C/EBP位点的结合模式存在明显差异。此外,我们证明C/EBP位点对于伪狂犬病病毒(PRV)ICP4抑制FIV LTR指导的基因表达是必需的。C/EBP位点足以赋予PRV ICP4对异源启动子的抑制作用。这些数据表明,FIV LTR中的C/EBP位点对于FIV基因表达和复制的正调控很重要,并且也是PRV ICP4对FIV基因表达进行负调控所必需的。