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首页> 外文期刊>Molecular and Cellular Biology >Regulation of RNA Polymerase I-Dependent Promoters by the Hepatitis B Virus X Protein via Activated Ras and TATA-Binding Protein
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Regulation of RNA Polymerase I-Dependent Promoters by the Hepatitis B Virus X Protein via Activated Ras and TATA-Binding Protein

机译：乙肝病毒X蛋白通过激活的Ras和TATA结合蛋白对RNA聚合酶I依赖性启动子的调控

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The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with anX gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838–6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the addition of recombinant TBP to S2 extracts stimulated rRNA promoter activity in vitro. Together, these results demonstrate that the HBV X protein up-regulates RNA Pol I-dependent promoters via a Ras-activated pathway in two distinct cell lines. The enhanced promoter activity can, at least in part, be attributed to the X- and Ras-mediated increase in cellular TBP, a limiting transcription component.
机译：乙型肝炎病毒（HBV）X蛋白对于病毒感染至关重要，证据表明，它是HBV介导的肿瘤发生的重要因素。 X已显示可激活多种依赖于RNA聚合酶（Pol）II以及依赖于RNA Pol III的启动子。在这项研究中，我们研究了X调节依赖于RNA Pol I的rRNA转录的可能性。在人类肝癌Huh7和果蝇 Schneider S2细胞系中，X表达均刺激rRNA启动子活性。从稳定表达了 X 基因的X表达细胞制备的提取物也具有增强的转录rRNA启动子的能力。通过确定该调节事件是否依赖于Ras激活和增加的TATA结合蛋白（TBP）水平来检查X反式激活的机制。我们以前的研究表明，X和Ras的激活会增加TBP的细胞水平（H.-D. Wang，A。Trivedi和DL Johnson，Mol。Cell。Biol。17：6838–6846 （1997年）。 Ras显性阴性形式的表达阻断了X介导的rRNA启动子的诱导，而Ras的组成型激活形式的表达模仿了X对rRNA启动子活性的增强作用。当在Huh7或S2细胞中过表达TBP时，观察到rRNA启动子活性呈剂量依赖性增加。为了分析TBP的增加是否间接调节rRNA启动子的活性，通过增加依赖于RNA Pol II的启动子的活性，构建了具有突变的<果>果蝇 TBP cDNA，从而消除了其刺激RNA Pol II的能力。依赖性启动子。 S2细胞中野生型TBP的瞬时表达增加了特定的RNA Pol I和Pol II依赖性启动子的活性。突变型TBP蛋白的表达未能增强依赖于RNA Pol II的启动子的活性，但该蛋白完全保留了其刺激rRNA启动子的能力。此外，在S2提取物中添加重组TBP可刺激rRNA启动子的体外活性。总之，这些结果表明，HBV X蛋白通过Ras激活的途径在两种不同的细胞系中上调RNA Pol I依赖性启动子。启动子活性增强可以至少部分归因于细胞TBP（限制性转录成分）的X-和Ras介导的增加。

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