Wu F K, Garcia J A, Harrich D, Gaynor R B
Department of Medicine, UCLA School of Medicine.
EMBO J. 1988 Jul;7(7):2117-30. doi: 10.1002/j.1460-2075.1988.tb03051.x.
Transcription of the human immunodeficiency virus type 1 (HIV-1) is regulated by viral proteins and cellular factors that bind to the viral long terminal repeat (LTR). At least five regions of the HIV LTR serve as binding sites for HeLa cellular proteins. One region containing two copies of the sequence GGGACTTTCC functions as an enhancer element for HIV transcriptional regulation. Another region between -17 and +44 known as the TAR region contains two copies of the sequence CTCTCTGG and is also important in tat-induced activation of the HIV LTR. HeLa cell extracts were used to purify cellular proteins binding to portions of the enhancer region (EBP-1) and the TAR region (UBP-1) by a combination of conventional and DNA affinity chromatography. Several species of proteins of between 55 and 60 kd were found to bind to specific sequences in the enhancer region and these proteins also bound to a portion of the NF-kappa B binding site in the immunoglobulin kappa enhancer. Two proteins of between 61 and 63 kd were the major species found to bind to specific sequences in the TAR region and fractions containing these proteins also bind to the TATA region. Both UBP-1 and EBP-1 exhibited specific binding as demonstrated by both UV cross-linking and DNase I footprinting. Mutations of either the enhancer or TAR regulatory regions prevented binding of these purified factors. These results demonstrate the binding of highly purified cellular proteins to important transcriptional regulatory regions in the HIV LTR.
人类免疫缺陷病毒1型（HIV-1）的转录受与病毒长末端重复序列（LTR）结合的病毒蛋白和细胞因子调控。HIV LTR的至少五个区域作为HeLa细胞蛋白的结合位点。一个含有两个GGGACTTTCC序列拷贝的区域作为HIV转录调控的增强子元件发挥作用。另一个位于-17至+44之间的区域称为TAR区域，含有两个CTCTCTGG序列拷贝，在tat诱导的HIV LTR激活中也很重要。通过常规色谱法和DNA亲和色谱法相结合，利用HeLa细胞提取物纯化与增强子区域部分（EBP-1）和TAR区域（UBP-1）结合的细胞蛋白。发现几种55至60kd的蛋白质与增强子区域的特定序列结合，并且这些蛋白质也与免疫球蛋白κ增强子中的NF-κB结合位点的一部分结合。两种61至63kd的蛋白质是在TAR区域与特定序列结合的主要蛋白质种类，含有这些蛋白质的组分也与TATA区域结合。UV交联和DNase I足迹法均表明UBP-1和EBP-1表现出特异性结合。增强子或TAR调控区域中的突变阻止了这些纯化因子的结合。这些结果证明了高度纯化的细胞蛋白与HIV LTR中重要转录调控区域的结合。
