Inhibition of high risk HPV31 E8^E2 repressor activity enables differentiation-independent genome amplification and E4 expression

Persistent infections with high-risk human papillomaviruses (HPV) can result in different malignancies. Productive replication of HPV is normally restricted to suprabasal keratinocytes that have entered terminal differentiation and is characterized by vegetative genome amplification, activation of the late promoter, and expression of the viral late E4 protein. Cells undergoing productive replication remain in a prolonged G2 phase and exit the cell cycle without division. The viral E8^E2 protein binds to NCoR/SMRT co-repressor complexes to repress viral transcription and replication in undifferentiated keratinocytes, but the biological rationale for this repression has remained unclear. Recent studies have revealed that Mus musculus PV1 E8^E2 prevents late viral E4 expression in undifferentiated cells to enable tumor formation in vivo. Here, we demonstrate that loss of E8^E2 function in high-risk HPV31 leads to inappropriate activation of genome amplification in undifferentiated keratinocytes, resulting in expression of E4 protein and cell cycle perturbation which explains why HPV31 E8^E2 mutant genomes fail to be maintained as episomes and instead are always found integrated in surviving cell lines. Interestingly, this is independent from E4 expression suggesting that vegetative genome amplification is sufficient to prevent cell division. Remarkably, depletion of NCoR/SMRT complexes in cell lines maintaining HPV31 episomes phenocopies E8^E2 inactivation and induces genome amplification and E4 expression. Notably, most E4-positive cells generated by E8^E2 inactivation or NCoR/SMRT depletion retain basal-like characteristics, indicating that genome amplification and E4 expression can be uncoupled from differentiation when E8^E2 repression is relieved. Interestingly, differentiation diminishes the effects of NCoR/SMRT depletion, suggesting that E8^E2 activity is likely inactivated during differentiation to permit productive replication. Collectively, these findings identify E8^E2 as a critical gatekeeper preventing premature genome amplification and E4 expression in basal keratinocytes and suggest that targeting the E8^E2-NCoR/SMRT interaction may represent a novel antiviral strategy.