Investigation of HPV pathogenesis in the head and neck using novel tonsil models

Human papillomavirus type 16 (HPV16) is present in >90% of HPV-driven head and neck squamous cell carcinomas (HNSCC) whereas HPV18, the second most common high-risk HPV type, is rarely detected. To understand HPV16 pathogenesis in the head and neck, we developed isogenic HPV16 and HPV18 episome-containing primary human tonsil keratinocytes models, which accurately recapitulate the molecular phenotype of the tonsils, the primary site of HPV-driven HNSCC. Differentiation in organotypic raft culture showed evidence of productive infection including viral DNA amplification and expression of viral late proteins. Long-read Nanopore RNA-sequencing was used to characterise and compare HPV16 and HPV18 transcriptome organisation in undifferentiated cells. These studies demonstrated higher activity of the major viral early promoter in HPV16 cells, contributing to higher expression of HPV16 E7 oncogenes. Further, HPV18 cells showed increased levels of transcripts encoding E8^E2, a repressor of early viral transcription, suggesting that HPV18 elicits greater E8^E2-mediated regulatory control of the early promoter than HPV16. Finally, short-read Illumina sequencing was performed to investigate virus-mediated transcriptional reprogramming of host transcriptomes. Here, HPV16 was shown to establish a more immunosuppressive environment than HPV18 by strongly downregulating activities of central transcriptional immune mediators including NF-kB, IFN-α, and IFN-γ, impairing expression of critical antiviral effector genes like CD40, CCL2, and IL1B. This could provide an essential survival advantage for HPV16-infected cells in the tonsils.