A critical role for E2-p53 interaction during the human papillomavirus 16 life cycle

Abstract

Human papillomaviruses (HPV) are causative agents in ano-genital and oral cancers; HPV16 is the most prevalent type detected in human cancers. The HPV16 E6 protein targets p53 for proteasomal degradation to facilitate proliferation of the HPV16 infected cell. However, in HPV16 immortalized cells E6 is predominantly spliced (E6*) and unable to degrade p53. Here we demonstrate that human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), and HPV16 positive oropharyngeal cancers, retain significant expression of p53. In addition, p53 levels can be increased in HPV16+ head and neck cancer cell lines following treatment with cisplatin. Introduction of full-length E6 into HFK+HPV16 resulted in attenuation of cellular growth (in hTERT immortalized HFK, E6 expression promoted enhanced proliferation). An understudied interaction is that between E2 and p53 and we investigated whether this was important for the viral life cycle. We generated mutant genomes with E2 unable to interact with p53 resulting in profound phenotypes in primary HFK. The mutant induced hyper-proliferation, but an ultimate arrest of cell growth; {beta}-galactosidase staining demonstrated increased senescence, and COMET assays showed increased DNA damage compared with HFK+HPV16 wild type cells. There was failure of the viral life cycle in organotypic rafts with the mutant HFK resulting in premature differentiation and reduced proliferation. The results indicate that the E2-p53 interaction is critical during the HPV16 life cycle, and that disruption of this interaction has anti-viral potential. We discuss potential mechanisms to explain these phenotypes. ImportanceHuman papillomaviruses are causative agents in around 5% of all cancers. There are currently no antivirals available to combat these infections and cancers, therefore it remains a priority to enhance our understanding of the HPV life cycle. Here we demonstrate that an interaction between the viral replication/transcription/segregation factor E2 and the tumor suppressor p53 is critical for the HPV16 life cycle. HPV16 immortalized cells retain significant expression of p53, and the critical role for the E2-p53 interaction demonstrates why this is the case. If the E2-p53 interaction is disrupted then HPV16 immortalized cells fail to proliferate, have enhanced DNA damage and senescence, and there is premature differentiation during the viral life cycle. Results suggest that targeting the E2-p53 interaction would have therapeutic benefits, potentially attenuating the spread of HPV16.