Thompson F J, Elder J, Neil J C
Department of Veterinary Pathology, University of Glasgow, Bearsden, U.K.
J Gen Virol. 1994 Mar;75 ( Pt 3):545-54. doi: 10.1099/0022-1317-75-3-545.
Nuclear protein binding sites in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV) were identified by the method of DNase I footprinting. Using nuclear protein extracts from a feline T lymphoma cell line, several discrete footprints were generated upstream of the transcriptional initiation site (-50 to -150). The specificity of protein binding was examined by competition with oligonucleotides representing consensus DNA binding sites for known transcription factors. Binding to AP-1 (-124) and ATF (-58) motifs was observed, with cross-competition between these sites. A strong footprint signal was also detected over a tandemly repeated C/EBP motif (-94, -86) and an adjacent weaker footprint was found to be specific for an NF1 motif (-72/-63). The effect on FIV LTR promoter activity of progressively deleting these nuclear factor binding sites was examined by linking LTR deletion mutants to the chloramphenicol acetyltransferase (CAT) gene. Deletion of the AP-1 site caused a 10- to 25-fold loss of CAT activity whereas deletion past the ATF site reduced activity virtually to background levels. The effects of deleting the C/EBP and NF1 sites were less marked and varied according to cell type. Transactivation of the LTR was assayed using constructs linked to a CAT reporter gene. The full-length FIV LTR was not significantly trans-activated. However, the expression of a deleted LTR construct lacking the AP-4/AP-1 site but retaining C/EBP and ATF sites was partially restored by co-infection with FIV or by co-transfection with an infectious molecular clone of FIV (FIV-PPR). These results show that host transcription factors responsive to cellular activation have a major role in regulating FIV expression, and suggest that virus-coded trans-activators acting through U3 may play a role in some cellular environments.
采用DNA酶I足迹法鉴定了猫免疫缺陷病毒（FIV）长末端重复序列（LTR）中的核蛋白结合位点。利用猫T淋巴瘤细胞系的核蛋白提取物，在转录起始位点上游（-50至-150）产生了几个离散的足迹。通过与代表已知转录因子共有DNA结合位点的寡核苷酸竞争，检测了蛋白质结合的特异性。观察到与AP-1（-124）和ATF（-58）基序的结合，这些位点之间存在交叉竞争。在串联重复的C/EBP基序（-94，-86）上也检测到一个强足迹信号，并且发现相邻的较弱足迹对NF1基序（-72/-63）具有特异性。通过将LTR缺失突变体与氯霉素乙酰转移酶（CAT）基因连接，研究了逐步删除这些核因子结合位点对FIV LTR启动子活性的影响。删除AP-1位点导致CAT活性损失10至25倍，而删除超过ATF位点则使活性几乎降至背景水平。删除C/EBP和NF1位点的影响不太明显，并且根据细胞类型而有所不同。使用与CAT报告基因连接的构建体测定LTR的反式激活。全长FIV LTR没有明显的反式激活。然而，通过与FIV共感染或与FIV的感染性分子克隆（FIV-PPR）共转染，部分恢复了缺失AP-4/AP-1位点但保留C/EBP和ATF位点的缺失LTR构建体的表达。这些结果表明，对细胞激活有反应的宿主转录因子在调节FIV表达中起主要作用，并表明通过U3起作用的病毒编码反式激活因子可能在某些细胞环境中起作用。
