Kumar K U, Devireddy L R, Tang S C, Pater A, Pater M M
Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.
J Neurochem. 1996 Aug;67(2):473-81. doi: 10.1046/j.1471-4159.1996.67020473.x.
The nuclear factor 1 (NF-1) motifs, NF-1 II/III, In the two 98-bp repeats of the transcription-regulatory region of JC virus (JCV), have a critical role in brain-specific transcription from the JCV early promoter-enhancer. In this study, the role of these motifs in transactivation of the JCV late promoter-enhancer (JCVL) was examined in differentiating glial P19 embryonal carcinoma cells. The expression of papovaviral large tumor antigen (T-Ag) in the glial cells was shown by double immunofluorescence assays. By using site-directed mutagenesis and in vivo assays, the two wild-type NF-1 II/III sites, but not the third site, were found to be essential for the transactivation of JCVL by JCV T-Ag. In vitro transcription assays confirmed this specific transactivation and the transactivation was abolished by T-Ag antibody. In electrophoretic mobility shift assays, expression of JCV T-Ag increased the binding of a factor(s) to the 98-bp repeat. T-Ag antibody abolished the increase of binding. Binding assays with oligonucleotides of NF-1 11/111 motifs showed that the increased binding specifically required the wild-type NF-1 II/III sequences and confirmed the requirement of T-Ag. To determine the region of T-Ag necessary for transactivation Of JCVL, the coding sequences were mutated. The amino-terminal region of JCV Ag in amino acids 1-437 was essentially required for efficient transactivation. These results indicated that transactivation of JCVL and increased binding require a factor(s) found specifically in glial cells, the JCV NF-1 II/III sites, and the T-Ag amino-terminal region.
核因子1（NF-1）基序，即NF-1 II/III，位于JC病毒（JCV）转录调控区的两个98bp重复序列中，在JCV早期启动子增强子的脑特异性转录中起关键作用。在本研究中，在分化的神经胶质P19胚胎癌细胞中检测了这些基序在JCV晚期启动子增强子（JCVL）反式激活中的作用。通过双重免疫荧光测定法显示了神经胶质细胞中乳头多瘤空泡病毒大肿瘤抗原（T-Ag）的表达。通过定点诱变和体内试验，发现两个野生型NF-1 II/III位点而非第三个位点对于JCV T-Ag反式激活JCVL至关重要。体外转录试验证实了这种特异性反式激活，并且T-Ag抗体消除了这种反式激活。在电泳迁移率变动试验中，JCV T-Ag的表达增加了一种因子与98bp重复序列的结合。T-Ag抗体消除了结合的增加。用NF-1 11/111基序的寡核苷酸进行的结合试验表明，结合增加特别需要野生型NF-1 II/III序列，并证实了对T-Ag的需求。为了确定反式激活JCVL所需的T-Ag区域，对编码序列进行了突变。JCV Ag氨基酸1-437的氨基末端区域对于有效的反式激活至关重要。这些结果表明，JCVL的反式激活和结合增加需要在神经胶质细胞中特异性发现的一种因子、JCV NF-1 II/III位点和T-Ag氨基末端区域。
