Identifying strategies to modulate the repression activity of the papillomavirus E8^E2 protein

• Applicant: Professor Dr. Frank Stubenrauch
• Subject Area: Virology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 578948872

Persistent infections with high-risk HPV, such as HPV16 and HPV31, are a major cause of anogenital and oropharyngeal carcinomas. HPV infect basal keratinocytes, where only early viral genes are expressed and genome replication remains limited. In suprabasal differentiating cells, however, genome amplification, late gene expression, abundant E4 production, and virion formation occur. These cells exit the cell cycle after an extended G2 phase and no longer proliferate. In addition to the replication factor E2, papillomaviruses encode the conserved fusion protein E8^E2, which acts as a potent repressor of viral transcription and replication through interaction with NCoR/SMRT corepressor complexes (NCoR/SMRT, TBL1/TBLR1, GPS2, HDAC3). Loss of E8^E2 or disruption of its binding to NCoR/SMRT leads to enhanced genome replication and gene expression in undifferentiated keratinocytes. Notably, this repression is independent of HDAC3, in contrast to cellular NCoR/SMRT target genes, suggesting a previously unknown, HPV-specific mechanism. Functional studies demonstrate that E8^E2 knockout or NCoR-binding–deficient HPV31 genomes induce genome amplification and E4 expression already in basal keratinocytes, thereby impairing episomal maintenance, immortalization in vitro, and tumor formation in vivo. Likewise, knock-down of NCoR/SMRT components in HPV31-positive cell lines triggers genome amplification and E4 expression in undifferentiated keratinocytes. These findings suggest that disruption of E8^E2 activity could force HPV into a productive replication cycle in basal cells, preventing persistence and thus oncogenesis. The E8^E2–NCoR/SMRT axis therefore represents an attractive antiviral target. To elucidate the underlying mechanisms, biotin proximity labeling in living cells will be employed. In contrast to co-immunoprecipitation, this approach identifies both direct and indirect interactors as well as viral DNA-associated proteins near E2/E8^E2 binding sites. Initial AlphaFold3 modeling and co-immunoprecipitation identify TBL1/TBLR1 as likely direct binding partners. Immunofluorescence analyses show that E2 accumulates in viral replication centers with nuclear foci phenotypes that vary depending on E8^E2 status. Organotypic HPV wild-type cultures predominantly display E8- and E8-NCoR-like phenotypes, suggesting that E8^E2 activity decreases during differentiation. The project aims to (i) identify proteins mediating HDAC3-independent repression of HPV by NCoR/SMRT complexes, (ii) characterize the interaction between E8^E2 and TBL1/TBLR1, and (iii) define mechanisms impairing E8^E2 activity in differentiated keratinocytes. In the long term, the goal is to assess whether targeted modulation of E8^E2 function can be exploited as an innovative anti-HPV strategy.

• DFG Programme: Research Grants