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.
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