REGULATION OF PAPILLOMAVIRUS EXPRESSION BY EPIDERMAL GROWTH FACTOR

这个项目的长期目标是定义细胞和分子 导致呼吸道乳头状瘤复发的机制。临床和临床 确定人乳头瘤病毒(HPV)感染的病理效应 HPV编码蛋白的表达。反过来，蛋白质的水平是 由病毒启动子的mRNAs转录调控。监管 HPV转录是一个复杂的过程，受 结合病毒上游调控区的病毒和细胞因子 (URR)。转录水平，以及特定的转录 是由宿主细胞的分化决定的， 对外部激素、生长因子和其他因素的反应 不确定的因素。我们发现，表皮生长因子(EGF)既有 抑制HPV11转录，诱导HPV11异常 分化是乳头状瘤的主要表型。这样做的目的是 提案是确定如何监管HPV11早期启动子，以及 检验EGF受体激活差异的假说 调节E6、E7和E1ORF的表达，部分通过 URR中的EGF响应元素。 具体目标是： L：检验表皮生长因子暴露改变相对转录的假说 从乳头状瘤细胞中的HPV6/11E6、E7和E1早期启动子，使用 核糖核酸酶保护试验。 2.确定HPV 11URR是否调节E7和E1的表达， 该规定是否需要病毒蛋白，以及是否涉及 URR中潜在的EGF响应消音器序列。这件事会做到的 利用显微注射或转染荧光素酶表达载体 单独的细胞，或与HPV11E1和E2的表达构建。 3.确定EGF是否诱导HPV表达下调和 细胞分化需要p21(Ras)和/或磷脂酰肌醇特异性 磷脂酶C-γ1。这将通过使用特定的抑制剂来实现 这两个第二信使，测量两个荧光素酶的表达 正常细胞和乳头状瘤细胞中的结构和HPV11转录本 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.