Negative feedback regulation of CHK2 in VIN18 raft cultures naturally infected with HPV18

The life cycle of human papillomavirus (HPV) is intricate, and a lack of appropriate in vitro models for natural HPV infection has led to a dearth of effective treatments for infection and related tumors. The HPV life cycle is strictly dependent on the differentiation of epithelial cells. Therefore, in our previous study, we used conditional reprogramming(CR) technology to establish human vaginal intraepithelial neoplasia cell infected with HPV18 naturally (VIN18) and verified that HPV18 completes its viral life cycle in these cells. This article refers to it as VIN18. In this study, we utilize VIN18 to establish a 3D differentiation model that facilitates a time gradient of infection within raft-like organotypic cultures.Our findings clarify previous understanding of the interaction between HPV18 and the host during the viral physiological cycle. We observed that under undifferentiated conditions, ATM-CHK2 is not essential for the genomic stability of HPV18. However, the differentiation environment primarily activates HPV18 amplification via ATM/CHK2 signaling. CHK2 exerts negative feedback regulation on the activity of upstream ATM. Moreover, upregulation of p53 and p21 leads to a reduction in cyclin D1. Consequently, increased HPV18 E7 expression induces the re-entry of VIN18 cells into S phase, resulting in elevated expression of cyclin A2 and cyclin B1, which causes the cell cycle arrest in S phase/G2 phase, thereby supporting viral genome amplification. This study provides a valuable new model for HPV biology research and offers insights into the regulation of the HPV life cycle through the differentiation process.