Xiao J, Buehring G C
School of Public Health, University of California, Berkeley, California 94720, USA.
J Virol. 1998 Jul;72(7):5994-6003. doi: 10.1128/JVI.72.7.5994-6003.1998.
Bovine leukemia virus (BLV) is a member of the human T-cell leukemia virus (HTLV)/BLV group of retroviruses. These viruses regulate their own transcription by producing Tax, a protein which activates the virus promoter region, the long terminal repeat (LTR). To explore the molecular mechanisms involved in the transactivation, we identified protein binding elements by in vivo footprinting and analyzed their function by site- directed mutagenesis. We used in vivo dimethyl sulfate footprinting by ligation-mediated PCR to detect constitutive in vivo protein-DNA interactions in a BLV-producing cell line, Bat2Cl6. The U3 region and part of the R region of the LTR were footprinted. In addition to the cis-acting elements (three cyclic AMP-responsive elements [CREs] and two AP4 sites) reported by others to be important for Tax-mediated activation of the BLV LTR, we found footprints in regions flanking these elements and in the core promoter region. The importance of these sites for transcriptional activation was studied by site-directed mutagenesis followed by promoter function analysis of the mutants with a chloramphenicol acetyltransferase reporter system. Our data corroborate those of others showing that the CREs are necessary for transactivation of the LTR, and they identify two new functional sites not previously reported by others. We show that the middle region of the BLV U3 contains multiple dual-functioning cis-acting elements which act as either positive or negative regulatory elements depending on the cell type tested. This is the first report of a functional mapping of the cis-acting elements of a virus of the HTLV/BLV group.
牛白血病病毒（BLV）是逆转录病毒中人类T细胞白血病病毒（HTLV）/BLV组的成员。这些病毒通过产生Tax蛋白来调节自身转录，Tax蛋白可激活病毒启动子区域，即长末端重复序列（LTR）。为了探究反式激活所涉及的分子机制，我们通过体内足迹法鉴定了蛋白质结合元件，并通过定点诱变分析了它们的功能。我们利用连接介导的PCR进行体内硫酸二甲酯足迹分析，以检测产生BLV的细胞系Bat2Cl6中组成型的体内蛋白质 - DNA相互作用。LTR的U3区域和部分R区域出现了足迹。除了其他人报道的对Tax介导的BLV LTR激活很重要的顺式作用元件（三个环磷酸腺苷反应元件[CREs]和两个AP4位点）外，我们在这些元件侧翼区域和核心启动子区域也发现了足迹。通过定点诱变，随后用氯霉素乙酰转移酶报告系统对突变体进行启动子功能分析，研究了这些位点对转录激活的重要性。我们的数据证实了其他人的研究结果，即CREs对LTR的反式激活是必需的，并且我们鉴定出了两个以前未被其他人报道的新功能位点。我们表明，BLV U3的中间区域包含多个双功能顺式作用元件，根据所测试的细胞类型，它们可作为正调控元件或负调控元件。这是HTLV/BLV组病毒顺式作用元件功能图谱的首次报道。
