HPV E6 inhibits E6AP to regulate epithelial homeostasis by modulating keratinocyte differentiation commitment and...

Area of Science:

• Cell Biology
• Virology
• Molecular Biology

Background:

• Human papillomaviruses (HPV) establish persistent infections by disrupting epithelial homeostasis.
• The E6 oncoproteins of HPV are key drivers of cellular dysregulation.
• Epithelial homeostasis involves intricate processes like cell density, cell cycle control, and differentiation.

Purpose of the Study:

• To elucidate the regulatory roles of E6AP and NHERF1, cellular targets of HPV E6 proteins, in maintaining epithelial homeostasis.
• To investigate how HPV11 and HPV16 E6 oncoproteins modulate keratinocyte phenotype and signaling pathways.
• To define the specific contributions of E6AP and NHERF1 interactions with E6 in HPV-induced cellular changes.

Main Methods:

• Utilized FUCCI and cell-cell competition assays to study keratinocyte behavior.
• Employed depletion and mutant expression strategies for E6AP and NHERF1.
• Conducted RNA sequencing to analyze transcriptional profiles and YAP target gene activation.
• Analyzed HPV-infected patient tissue (condyloma) and organotypic raft cultures.

Main Results:

• Depletion of E6AP or expression of HPV11/16E6 increased keratinocyte density, cell cycle activity, and delayed differentiation.
• HPV11 and 16 infected tissues showed increased cell density and delayed differentiation, mirroring experimental findings.
• E6AP and NHERF1 were reduced in HPV11 condyloma tissue compared to uninfected epithelium.
• Loss of HPV11 E6/E6AP binding abolished homeostasis regulation; loss of E6/NHERF1 binding altered differentiation thresholds.
• RNA sequencing revealed YAP target gene induction and differentiation gene downregulation in E6-expressing and E6AP-/- cells.
• HPV11 E6 activated Yap in various systems, with E6AP and NHERF1 playing crucial roles.

Conclusions:

• Alpha group HPV E6 proteins, through conserved interactions with E6AP, modulate keratinocyte phenotype and epithelial homeostasis.
• These interactions lead to alterations in downstream pathways, including those involving NHERF1 and YAP.
• E6AP is essential for HPV16 E6 homeostasis functions, while NHERF1's role is more nuanced depending on the specific E6 protein.
• The study provides a model for how HPV E6 proteins hijack cellular machinery to promote persistent infection.