ABSTRACT 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 its 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 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. IMPORTANCE Human 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) as 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.
James et al. (Mon,) studied this question.