Virus-host interaction: investigating novel transcription factors involved in coupling HPV life cycle and epithelial differentiation.

High-risk human papillomavirus (HPV) causes almost all cervical cancer, and HPV-18 is the second most prevalent type. The HPV life cycle is intimately associated with the epithelial differentiation program, and the repertoire of cellular transcription factors (TFs) has a crucial role in coordinating viral gene expression across epithelial layers. We aimed to identify host TFs involved in the virus differentiation-dependent life cycle. We initially compared the DNA binding activity of 345 TFs in nuclear extracts of undifferentiated and high calcium-induced differentiated HaCaT cells. Next, we searched in silico for putative binding sites within the HPV-18 long control region (LCR) for the TFs affected by differentiation. Chromatin immunoprecipitation assays (ChIP) were used to assess direct binding of selected TFs, protein levels were evaluated by immunohistochemistry in rafts obtained from human foreskin keratinocytes (HFK) and HPV-18 E6/E7 immortalized HFK, and the impact upon LCR activity was measured in reporter assays. The binding activity of 40 TFs was influenced by differentiation among which 16 putative binding sites within the LCR were identified. We next focused our analysis in PAX6, HMGB1, FOXI1, and NFE2, and all bound to the LCR except FOXI1. Whether FOXI1 activates HPV-18 early promoter activity, PAX6, HMGB1, and NFE2 inhibit viral transcription in undifferentiated normal HFK in which these proteins are mostly expressed. Finally, HPV-18 E6/E7 impaired the normal expression of these TFs throughout epithelial layers. In conclusion, we describe the involvement of PAX6, HMGB1, NFE2, and FOXIl in coupling HPV-18 transcriptional regulation with the epithelia differentiation program, a key event for the productive HPV life cycle. Furthermore, these TFs may also be relevant for HPV-driven carcinogenesis. The identification of proteins involved in the differentiation-dependent HPV life cycle and viral induced transformation may contribute to unveiling novel therapeutic targets for HPV-related malignancies. Author SummaryHPV-16 and HPV-18 respond together for over 70% of cervical cancers. HPV infection is established in basal layers cells of the stratified epithelia and the viral life cycle is highly dependent on the epithelial differentiation program. In this study, we focused on identifying host transcription factors (TFs) involved in the finely tuned regulation of the HPV-18 differentiation-dependent life cycle. We initially screened 345 cellular TFs and found 40 with differential binding activity after differentiation, among which we chose to focus on PAX6, HMGB1, FOXI1, and NFE2. Besides investigating TFs binding to HPV-18 LCR in silico and with immunoprecipitation assays, we evaluated protein levels throughout epithelial layers and demonstrated that the impact of these TFs upon HPV-18 early promoter activity depends on the differentiation status of cells. Finally, we show that HPV-18 oncoproteins lead to abnormal expression of these TFs. Our results contribute to a clearer understanding of pivotal mechanisms governing spatiotemporal regulation of viral early transcription and open avenues for innovative therapeutic strategies for HPV-associated diseases.