Analysis of HPV-18 transcriptional activity during cell differentiation

The human papillomavirus (HPV) life cycle is tightly linked to the keratinocytes differentiation program. The repertoire of transcription factors (TFs) expressed in the host cell has a crucial role in coordinating viral gene expression throughout different layers of the stratified squamous epithelium. In turn, the expression of viral oncogenes modifies the cellular environment culminating ultimately in cell tumorigenic transformation. The main purpose of this study was to better understand the functional role of the host TFs composition in assisting the virus to complete the life cycle during infection. First, we compared the level of 345 TFs in undifferentiated and differentiated keratinocytes and found the differential expression of 30 TFs. Since TFs that have binding sites within the viral long control region (LCR) have a potentially stronger influence on viral transcriptional regulation, we search for putative binding sites for the 30 TFs within HPV-18 LCR with in silico tools and then confirmed the binding of selected TFs with qPCR-ChiP assays. Therefore, we identified four novel TFs binding sites within the LCR: HMGB1, PAX-6, FOXI1 e NF-E2. Further, the functional approach revealed that the transient co-transfection of each of these four TFs with a construct of pGL3-18LCR was able to impact the HPV-18 early promoter in a dose-dependent manner. In addition, we observed a differential regulation upon the promoter activity regarding the differentiation state of the keratinocytes. Finally, it was demonstrated that the HPV-18 oncoproteins expression leads to an increase in the levels of these factors. Additionally, their expression is variable according to the cell transformation. Therefore, we identified four new cellular TFs that may be potentially relevant for the regulation of HPV transcription during differentiation and malignant transformation. Expanding this knowledge contributes to a better understanding of the biology and pathogenesis of diseases associated with HPV-18 (AU)