The role of E6 target proteins in the maintenance of the HPV transformed phenotype

Farrukh, Sannia (2025). The role of E6 target proteins in the maintenance of the HPV transformed phenotype. Doctor of Philosophy (PhD) thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00102169

Abstract

Human papillomavirus (HPV)-driven cancers are highly prevalent worldwide. During infection, the HPV E7 oncoprotein promotes S-phase entry, increasing cellular DNA replication. Normally, this would result in p53-induced growth arrest or apoptosis, but the high-risk HPV E6 oncoprotein forms a complex with the ubiquitin ligase E6AP to target p53 for degradation through the ubiquitin-proteasome pathway. However, some residual p53 is detectable in many HPV-positive cells and lesions. To investigate the role of this residual p53, we generated an HPV-positive p53-knockout (KO) HeLa cell line, which exhibited reduced growth and metabolic activity, especially under nutrient-limited conditions. Transcriptomic analysis of these cells revealed gain-of-function (GOF) p53 activities and enrichment of wild-type p53 pathways. Functional assays showed that their survival, invasion, and migration abilities were reduced, while immunoprecipitation analysis confirmed a pool of GOF p53. These findings suggest a role for residual p53 in HPV-positive cancers and raise questions about the functional significance of the E6-p53 interaction.

The E6-E6AP interaction is critical for E6 stability: silencing E6AP in HPV-positive cells rescues p53 but reduces E6 levels. Interestingly, E6 can degrade certain substrates independently of E6AP. We therefore introduced a catalytically inactive E6AP C843A mutation into HeLa cells, blocking degradation of targets like p53 and Scribble without affecting cell proliferation and survival. Our findings suggest that E6AP’s catalytic activity may not be essential for HPV-positive cell growth.

Additionally, we conducted proteomic analyses on differentiated and undifferentiated cells. By reprogramming HaCaT cells into induced pluripotent stem cells, we studied changes in HPV E6-PDZ interactions across the viral life cycle. Proteomic studies on papillomaviruses from various species highlight evolutionarily conserved mechanisms of HPV-induced carcinogenesis.

Using gene-editing techniques, we have produced these important model cell systems that can address major questions about the HPV life cycle and HPV’s role in inducing certain highly prevalent cancers.