HPV E6 inhibits E6AP to regulate epithelial homeostasis by modulating keratinocyte differentiation commitment and YAP1 activation

Wen Yin, Nagayasu Egawa, Ke Zheng, Heather Griffin, Pu Tian, Ademola Aiyenuro, Jacob Bornstein, John Doorbar

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Human papillomaviruses (HPV) cause persistent infections by modulating epithelial homeostasis in cells of the infected basal layer. Using FUCCI and cell-cell competition assays, we have identifed regulatory roles for E6AP and NHERF1, which are the primary HPV11 E6 cellular targets, as well as being targets of the high-risk E6 proteins, in processes governing epithelial homeostasis (i.e. cell density, cell cycle entry, commitment to differentiation and basal layer delamination). Depletion of E6AP, or expression of HPV11 or 16E6 increased keratinocyte cell density and cell cycle activity, and delayed the onset of differentiation; phenotypes which were conspicuously present in HPV11 and 16 infected patient tissue. In line with proposed E6 functions, in HPV11 condyloma tissue, E6AP and NHERF1 were significantly reduced when compared to uninfected epithelium. In experimental systems, loss of HPV11 E6/E6AP binding abolished 11E6’s homeostasis regulatory functions, while loss of E6/NHERF1 binding reduced the cell density threshold at which differentiation was triggered. By contrast, a NHERF1-binding mutant of 16E6 was not compromised in its homeostasis functions, while E6AP appeared essential. RNA sequencing revealed similar transcriptional profiles in both 11 and 16E6-expressing cells and E6AP-/- cells, with YAP target genes induced, and keratinocyte differentiation genes being downregulated. HPV11 E6-mediated Yap activation was observed in 2D and 3D (organotypic raft) cell culture systems and HPV-infected lesions, with both NHERF1, which is a regulator of the Hippo and Wnt pathways, and E6AP, playing an important role. As the conserved binding partner of Alpha group HPV E6 proteins, the precise role of E6AP in modulating keratinocyte phenotype and associated signalling pathways has not previously been defined. Our study suggests a model in which the preserved functions of the low and high-risk Alpha E6 proteins modulate epithelial homeostasis via E6AP activity, and lead to alteration of multiple downstream pathways, including those involving NHERF1 and YAP.

Original languageEnglish
Article numbere1011464
JournalPLoS Pathogens
Volume19
Issue number6 June
DOIs
StatePublished - Jun 2023

Bibliographical note

Publisher Copyright:
Copyright: © 2023 Yin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding

This work was supported by CSC Cambridge International Scholarship (ref. 303862057 to WY) and the UK Medical Research Council (grant ref. MC_PC_13050 and MR/S024409/1 to JD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

FundersFunder number
Medical Research CouncilMC_PC_13050, MR/S024409/1
Commonwealth Scholarship Commission303862057

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