Bridy, P. V. | bioRxiv Science

bioRxiv2025

Differentiation-Dependent Proximity Proteomics Identifies Novel Host Factors Linked to HPV16 E2 Function

Human papillomavirus 16 (HPV16) is a causative agent of oropharyngeal, cervical and anogenital cancers. The viral E2 protein is essential for viral genome replication, transcriptional regulation, episome maintenance, and activation of the host DNA damage response. Despite its central role, the full network of HPV16 E2 interactions with host proteins remains incompletely defined, particularly under differentiating conditions which support the complete viral life cycle. In this study, we used TurboID-based proximity labeling to characterize the interactome of HPV16 E2 and known host partner protein TOPBP1, in both undifferentiated monolayer and differentiating keratinocytes. We generated stable keratinocyte lines expressing doxycycline-inducible TurboID-tagged HPV16 E2 and confirmed that the tagged protein retained transcriptional, replicative, and DNA damage-inducing functions. Mass spectrometry analysis of streptavidin-enriched proteins identified both known and novel E2-associated host factors, including chromatin regulators, DNA repair proteins, and nucleolar components. Comparative analysis revealed a substantial overlap between E2 and TOPBP1 interactomes, and in situ validation by proximity ligation assay identified nucleolin (NCL) as a differentiation-dependent factor whose interaction with E2 is stabilized by TOPBP1. Functional studies demonstrated that NCL is required for episomal genome maintenance, highlighting a cooperative E2-TOPBP1- NCL axis critical for viral genome stability during differentiation. These findings provide a comprehensive view of the E2-associated protein landscape in stratified epithelial cells and reveal a mechanistic pathway through which HPV16 co-opts host factors to support genome maintenance, productive replication, and persistence. ImportanceHuman papillomaviruses (HPVs) establish persistent infections in stratified epithelia and rely on host DNA damage and repair factors to support their replication. The E2 protein is central to viral genome replication and maintenance, and depends heavily on its interaction with the host factor TOPBP1 for these functions. Here, we define the E2 and TOPBP1 interactomes in differentiating keratinocytes, and identify nucleolin (NCL) a critical differentiation- and TOPBP1-dependent E2 partner required for episomal genome stability. These findings expand the understanding of how HPV16 coordinates viral replication with host chromatin and DNA repair networks, uncovering a cooperative E2-TOPBP1-NCL axis that may represent a new target for antiviral intervention.

cancer biology↗

bioRxiv2025

E2 displacement of CIP2A from TOPBP1 activates the DNA damage response during papillomavirus life cycles

The papillomavirus life cycle is intricately linked to epithelial differentiation, and the virus manipulates the differentiation process to facilitate viral production. One such manipulation is activation of the DNA damage response (DDR) which promotes viral replication via homologous recombination. This report demonstrates that the papillomavirus transcription/replication/segregation factor E2 activates the DNA damage response (DDR). During differentiation, E2 displacement of CIP2A from TOPBP1 causes CIP2A to bind and inhibit PP2A resulting in DDR activation via ATM phosphorylation. The DDR promotes inhibitory interaction of DBC1 with the class III deacetylase SIRT1, which further boosts the DDR via increased acetylation and stability of viral and host proteins. E2 forms a complex with TOPBP1 and ATM, while preventing ATR activation by blocking TOPBP1-ATR interaction. This "ATM up ATR down" phenotype promotes viral replication via ATM promotion of homologous recombination, and cell proliferation via inhibition of ATR. We demonstrate this mechanism of DDR activation in multiple systems: keratinocytes expressing only E2, in foreskin keratinocytes immortalized by HPV16, in HPV16 positive keratinocytes derived from a cervical lesion, in pre-neoplastic lesions induced by mouse papillomavirus MmuPV1, in head and neck cancer cell lines that retain E2 expression, and in HPV16 positive oropharyngeal patient derived xenografts that retain E2 expression. ATM inhibition preferentially killed cells expressing E2, presenting a novel strategy for treating HPV early preneoplasia and a large subset of HPV+ oropharyngeal cancers retaining E2 expression and episomal genomes.

cancer biology↗