Van den Elsen P, Houweling A, Van der Eb A
Virology. 1983 Jul 30;128(2):377-90. doi: 10.1016/0042-6822(83)90264-7.
Previous work has suggested that morphological transformation of cultured cells by human adenoviruses of subgroups A, B, and C is predominantly a function of early region 1b (E1b), and that region E1a has a role in immortalization. To test the hypothesis that region E1b is essentially responsible for the induction of the transformed phenotype the transforming activity of region E1b in the absence of region E1a was reinvestigated. In agreement with previous results, region E1b had no detectable transforming activity in primary baby rat kidney (BRK) cells nor in established rat cell lines. Since recent experimental evidence indicates that expression of E1b is blocked by a cellular factor which is inactivated by region E1a products, the regulatory signals in front of the coding sequence of region E1b were removed and replaced by the early promoter of SV40. These E1b-SV40pr plasmids had no detectable transforming activity in primary BRK cells, but they transformed normally in the presence of region E1a plasmids, demonstrating that both subregions are required for complete transformation of primary BRK cells. Transfection of the established rat cell line 3Y1 with the E1b-SV40pr plasmids did not result in complete morphological transformation either. Cotransfection of 3Y1 cells with E1b-SV40pr plasmids and pAG60 (a plasmid which harbors the kanamycin-resistance gene of Tn5) resulted in the appearance of foci of cells resistant to the antibiotic G-418. These colonies expressed the region E1b polypeptides to levels comparable to those found in cells transformed with intact region E1. Despite the presence of the E1b proteins the cells appeared essentially untransformed, in contrast to foci obtained after cotransfection of 3Y1 cells with mixtures of p5XhoI C (comprising region E1 DNA) and pAG60. These results indicate that complete transformation is a function of both regions E1a and E1b and that region E1a must have an important role in morphological transformation.
先前的研究表明，A、B和C亚组的人腺病毒对培养细胞的形态转化主要是早期区域1b（E1b）的作用，而区域E1a在细胞永生化过程中发挥作用。为了验证E1b区域本质上负责诱导转化表型这一假说，我们重新研究了在没有E1a区域的情况下E1b区域的转化活性。与先前的结果一致，E1b区域在原代新生大鼠肾（BRK）细胞和已建立的大鼠细胞系中均未检测到转化活性。由于最近的实验证据表明E1b的表达被一种细胞因子阻断，而该细胞因子会被E1a产物灭活，因此我们去除了E1b编码序列前的调控信号，并用SV40的早期启动子进行替换。这些E1b - SV40pr质粒在原代BRK细胞中未检测到转化活性，但在存在E1a区域质粒的情况下能够正常转化，这表明两个亚区域对于原代BRK细胞的完全转化都是必需的。用E1b - SV40pr质粒转染已建立的大鼠细胞系3Y1也未导致完全的形态转化。将3Y1细胞与E1b - SV40pr质粒和pAG60（一种携带Tn5卡那霉素抗性基因的质粒）共转染，导致出现对抗生素G - 418有抗性的细胞集落。这些集落中E1b区域多肽的表达水平与用完整E1区域转化的细胞中发现的水平相当。尽管存在E1b蛋白，但与用p5XhoI C（包含E1区域DNA）和pAG60混合物共转染3Y1细胞后获得的集落相比，这些细胞基本未发生转化。这些结果表明，完全转化是E1a和E1b两个区域共同作用的结果，并且E1a区域在形态转化中必定起着重要作用。
