Kern F G, Pellegrini S, Cowie A, Basilico C
J Virol. 1986 Oct;60(1):275-85. doi: 10.1128/JVI.60.1.275-285.1986.
To assess the effect of the polyomavirus (Py) early proteins, the large T (LT), middle T (MT), and small T (ST) antigens, on gene expression from the Py late promoter, replication-defective plasmid constructs with the bacterial chloramphenicol acetyltransferase (cat) gene linked to this promoter were cotransfected into mouse or rat cells with plasmids capable of producing either LT, MT, or all three early proteins. When target CAT plasmids contained a truncated early region and thus had the coding potential for MT and ST, base-line CAT activities were low, whereas cotransfection with an LT plasmid resulted in up to 70-fold stimulation of CAT activity that was also reflected in similar increases in the level of steady-state mRNA. Studies with target plasmids with deletions within the Py regulatory region indicated that at least the major LT-binding site C and a functional enhancer region were both required for maximal stimulation of CAT activity. However, although enhancer deletions totally suppressed the ability of target plasmids to be trans activated, a consistent two- to fourfold stimulation of CAT activity by LT was still observed with a plasmid in which all three major LT-binding sites were deleted. Of four mutant LTs incapable of binding Py DNA but retaining immortalization potential, only one showed a low but significant trans-activating ability. When the early coding region was completely eliminated from the target plasmid, base-line CAT activity was increased 10-fold. LT failed to stimulate CAT activity to the same levels observed with target plasmid containing the truncated early region, but this limited response could be enhanced by supplying, in addition, MT and ST. Our results suggest that LT trans activation may involve the formation of a complex of transcriptional factors which interacts with the enhancer, an interaction that is facilitated both by the binding of LT to the Py regulatory region and by the presence of MT or ST or both, and that a significant portion of LT stimulation of late gene expression is a result of the removal of the competing early transcriptional unit via autoregulation. In addition, our results suggest that LT trans activation involves a second indirect component acting independently of LT binding and that the immortalization and trans activation functions of LT can be dissociated.
为了评估多瘤病毒（Py）早期蛋白，即大T（LT）、中T（MT）和小T（ST）抗原，对Py晚期启动子基因表达的影响，将带有与该启动子相连的细菌氯霉素乙酰转移酶（cat）基因的复制缺陷型质粒构建体，与能够产生LT、MT或所有三种早期蛋白的质粒共转染到小鼠或大鼠细胞中。当靶标CAT质粒包含截短的早期区域，因而具有编码MT和ST的潜力时，基础CAT活性较低，而与LT质粒共转染导致CAT活性最多增加70倍，这也反映在稳态mRNA水平的类似增加上。对Py调控区域内有缺失的靶标质粒的研究表明，至少主要的LT结合位点C和一个功能性增强子区域对于最大程度刺激CAT活性都是必需的。然而，尽管增强子缺失完全抑制了靶标质粒被反式激活的能力，但对于一个缺失了所有三个主要LT结合位点的质粒，仍观察到LT对CAT活性有持续的两到四倍的刺激。在四个不能结合Py DNA但保留永生化潜力的突变LT中，只有一个表现出低但显著的反式激活能力。当从靶标质粒中完全去除早期编码区域时，基础CAT活性增加了10倍。LT未能将CAT活性刺激到与含有截短早期区域的靶标质粒所观察到的相同水平，但通过额外提供MT和ST，这种有限的反应可以得到增强。我们的结果表明，LT反式激活可能涉及转录因子复合物的形成，该复合物与增强子相互作用，LT与Py调控区域的结合以及MT或ST或两者的存在都促进了这种相互作用，并且LT对晚期基因表达的显著刺激部分是通过自调控去除竞争性早期转录单元的结果。此外，我们的结果表明，LT反式激活涉及第二个独立于LT结合起作用的间接成分，并且LT的永生化和反式激活功能可以分离。
