REGULATION OF PAPILLOMAVIRUS EXPRESSION BY EPIDERMAL GROWTH FACTOR

这个项目的长期目标是确定细胞和分子 导致复发性呼吸道乳头状瘤的机制。临床和 确定人乳头瘤病毒（HPV）感染的病理效应 通过表达HPV编码的蛋白质。反过来，蛋白质的水平是 受来自病毒启动子的mRNA转录的调节。调控 HPV的转录是一个复杂的过程，由以下因素的相互作用控制： 与病毒上游调控区结合的病毒和细胞因子 （URR）。转录水平，以及特定的转录本， 产生的，由宿主细胞的分化决定， 对外部激素和生长因子的反应和其他尚未 不确定的因素。表皮生长因子（EGF） 抑制HPV 11的转录并诱导异常的 分化是主要的乳头状瘤表型。这个目标 建议是确定HPV 11早期启动子是如何调节的， 测试EGF受体的激活差异 调节E6、E7和E1 ORF的表达，部分通过 URR中的EGF响应元件。 具体目标是： L.测试EGF暴露改变相对转录的假设 从乳头状瘤细胞中的HPV 6/11 E6、E7和E1早期启动子，使用 RNA酶保护测定。 2.确定HPV 11 URR是否调节E7和E1的表达， 该调节是否需要病毒蛋白，以及它是否涉及 URR中潜在的EGF应答沉默序列。为此将 使用荧光素酶表达构建体显微注射或转染到 细胞，或与HPV 11 E1和E2的表达构建体。 3.确定EGF诱导的HPV表达下调和 细胞分化需要p2 L（ras）和/或磷酸肌醇特异性 磷脂酶C-γ 1。这将与特定的抑制剂， 这两个第二信使，测量荧光素酶表达， 在正常和乳头状瘤细胞中的构建体和在正常和乳头状瘤细胞中的HPV 11转录物， 乳头状瘤细胞

The long term goal of this project is to define the cellular and molecular mechanisms that cause recurrent respiratory papillomas. The clinical and pathologic effects of Human Papillomavirus (HPV) infection are determined by expression of HPV encoded proteins. In turn, the level of protein is regulated by transcription of mRNAs from the viral promoters. Regulation of HPV transcription is a complex process, controlled by interactions of viral and cellular factors binding to the viral upstream regulatory region (URR). Level of transcription, and the specific transcripts that are produced, are determined by the differentiation of the host cell and response to external hormones and growth factors and other as yet undefined factors. We have found that epidermal growth factor (EGF) both suppresses transcription of HPV 11 and induces the abnormal differentiation that is the major papilloma phenotype. The goal of this proposal is to determine how the HPV 11 early promoters are regulated, and test the hypothesis that activation of the EGF receptor differentially modulates expression of the E6, E7 and E1 ORFs, mediated in part through an EGF-responsive element in the URR. The Specific Aims are: l. Test the hypothesis that EGF exposure alters relative transcription from the HPV 6/11 E6, E7 and E1 early promoters in papilloma cells, using RNase protection assays. 2. Determine whether the HPV 11 URR regulates expression of E7 and E1, whether the regulation requires viral protein, and whether it involves a potential EGF-responsive silencer sequence in the URR. This will be done using luciferase expression constructs microinjected or transfected into cells alone, or with expression constructs for HPV 11 E1 and E2. 3. Determine whether EGF-induced down-regulation of HPV expression and cell differentiation requires p2l(ras) and/or phosphoinositide-specific phospholipase C-gamma1. This will be done with specific inhibitors for these two second messengers, measuring both luciferase expression constructs in normal and papilloma cells and HPV 11 transcripts in papilloma cells.