Virus–Host Interaction: Investigating Novel Transcription Factors Involved in Coupling Human Papillomavirus Life Cycle and Epithelial Differentiation

High‐risk human papillomavirus (HPV) is the etiological agent of nearly all cervical cancers, with HPV‐18 being the second most prevalent type. The HPV life cycle is closely associated with epithelial differentiation, a process governed by cellular transcription factors (TFs). During progression toward malignancy, HPV disrupts differentiation and promotes uncontrolled cell proliferation. We aimed to identify differentiation‐modulated TFs linking viral transcription to differentiation, providing insights into mechanisms driving viral life cycle and pathogenesis. DNA‐binding activity of 345 TFs was compared between undifferentiated and differentiated keratinocytes to identify differentiation‐associated TFs. In silico analyses identified putative binding sites for these TFs within the HPV‐18 long control region (LCR). Chromatin immunoprecipitation confirmed direct binding of PAX6, HMGB1, and NFE2 to the LCR, but not FOXI1. Immunohistochemistry in keratinocyte raft cultures demonstrated differentiation‐dependent expression patterns for all four TFs. Functional assays revealed that each TF is capable of modulate HPV‐18 early promoter activity, with effects varying by differentiation status. Notably, expression of these TFs was disrupted by the viral oncoproteins E6 and E7, underscoring a mechanism by which HPV alters host differentiation. These findings identify novel differentiation‐linked regulators of HPV‐18 transcription and highlight host targets exploited by the virus in its life cycle and pathogenesis.