Shivakumar C V, Das G C
Department of Epidemiology, University of Texas Health Center at Tyler, Tex., 75710, USA.
Intervirology. 1998;41(2-3):103-9. doi: 10.1159/000024921.
The A enhancer of the polyomavirus early promoter is a 110-bp domain located in the late region and it contains the major late RNA initiation site. The 'core' of this enhancer binds several cellular proteins, including proteins PEA1, PEA2 and PEA3. Another element, NF-D/YY1, is also located in this enhancer. The A enhancer is known to stimulate the early promoter, contains auxiliary elements for replication and serves as the initiator for late transcription. It may also be involved in early-to-late switch. We were interested in investigating how the A-core- and NF-D-binding proteins regulate early and late promoter activity under nonreplicating conditions and how the protein-protein interactions affect the function of the individual elements. By point mutational analysis, we show that, except for PEA1, all other proteins activate the early and late promoters differentially under nonreplicating conditions. All three core-binding proteins, and the protein bound to the NF-D site, are activators and have a combinatorial effect on early promoter activity. On late transcription, only PEA1 acts positively and inactivation of the NF-D site is without any effect. In contrast, PEA2 and PEA3 have a repressor-like activity under nonreplicating conditions, indicating that these two proteins might be involved in repressing late transcription, probably early in infection. By increasing the spacing between two consecutive elements, we further show that protein-protein interaction is important for enhancer function. Transactivation of the early promoter was affected by mutations in all four protein-binding sites. Responsiveness of these factors in regard to the late promoter was parallel to their intrinsic promoter strength. The effects of middle and large T antigens are parallel for both the early and the late promoter, suggesting that the pathway(s) for transactivation function of these oncoproteins may overlap downstream. This study with cloned viral promoter will be reflective of situations in vivo, at least partially, under nonreplicating conditions.
多瘤病毒早期启动子的A增强子是一个位于晚期区域的110碱基对结构域，它包含主要的晚期RNA起始位点。该增强子的“核心”结合几种细胞蛋白，包括PEA1、PEA2和PEA3蛋白。另一个元件NF-D/YY1也位于此增强子中。已知A增强子可刺激早期启动子，包含复制辅助元件并作为晚期转录的起始子。它也可能参与早期到晚期的转换。我们感兴趣的是研究在非复制条件下，A核心结合蛋白和NF-D结合蛋白如何调节早期和晚期启动子活性，以及蛋白质-蛋白质相互作用如何影响各个元件的功能。通过点突变分析，我们发现，除了PEA1，所有其他蛋白在非复制条件下对早期和晚期启动子的激活作用不同。所有三种核心结合蛋白以及与NF-D位点结合的蛋白都是激活剂，对早期启动子活性具有协同作用。在晚期转录中，只有PEA1起正向作用，NF-D位点的失活没有任何影响。相反，PEA2和PEA3在非复制条件下具有类似阻遏物的活性，表明这两种蛋白可能参与抑制晚期转录，可能在感染早期。通过增加两个连续元件之间的间距，我们进一步表明蛋白质-蛋白质相互作用对增强子功能很重要。早期启动子的反式激活受到所有四个蛋白结合位点突变的影响。这些因子对晚期启动子的反应性与其内在启动子强度平行。中T抗原和大T抗原对早期和晚期启动子的作用是平行的，这表明这些癌蛋白的反式激活功能途径可能在下游重叠。这项对克隆病毒启动子的研究至少在部分非复制条件下将反映体内情况。
