NFX1-123 is required for keratinocyte differentiation and HPV 16 DNA maintenance in early and persistent infection models

Persistent high-risk human papillomavirus (HR HPV) infections cause nearly 5% of cancers worldwide, including cervical and oropharyngeal cancers. HR HPVs sustain long-term infections in stratified epithelial tissues by maintaining copies of viral genomes in basal keratinocytes and utilizing the cellular differentiation processes in the upper layers to coordinate key steps in the viral life cycle. We have demonstrated previously that the endogenous host protein NFX1-123 bound directly to the HR HPV type 16 oncoprotein E6 (16E6), and that this partnership increased keratinocyte proliferation and differentiation, two processes that are both required for persistent HPV 16 infections. As there is a range of endogenous expression of NFX1-123 in normal basal keratinocytes, we aimed to investigate the role of NFX1-123 abundance on the initiation and persistence of HPV 16 infections. In this study, we utilized keratinocytes with overexpressed, endogenous, or reduced levels of NFX1-123 and infected these cells with an HPV 16 quasivirus (Qsv) to interrogate changes to keratinocyte growth and viral DNA copy numbers with NFX1-123 modulation. Keratinocytes with reduced NFX1-123 had blunted differentiation and stratification in raft cultures, and this correlated with decreased HPV 16 Qsv DNA copies. Similarly, when we reduced NFX1-123 in W12E cells, a cervical cell line harboring episomal HPV 16 genomes, we saw a decrease in raft culture differentiation and thickness, and fewer viral genome copies compared to W12E cells with endogenous NFX1-123. These findings indicate the importance of NFX1-123 on the initiation and maintenance of HPV 16 infections that may persist and progress to cancer.IMPORTANCEA high-risk human papillomavirus (HPV) infection that persists for decades is the biggest risk factor for the development of cervical and other HPV-associated cancers. The ability of HPV to maintain its genome and persist in stratified epithelium is dependent on its partnership with host proteins and its co-opting of cellular pathways. Here, we investigate how the abundance of HPV 16's host protein partner NFX1-123 affects viral genome maintenance and replication in monolayer and three-dimensional cultures, modeling both early and persistent infection. This study reveals that NFX1-123 is required for keratinocyte differentiation and stratification in raft cultures, and the disruption of these processes significantly impedes the ability of HPV 16 to maintain its episomal genome at consistent copy numbers. These findings indicate the importance of NFX1-123 for the persistence of HPV 16 infections and encourage further investigation of NFX1-123 as a potential therapeutic target in HPV 16-positive cancers and precancers.