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Investigating Early Lesion Formation Following Papillomavirus Infection Using a Mouse Model and Cell Culture



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Saunders-Wood, Taylor 


Papillomaviruses (PV) are small non-enveloped double-stranded DNA tumour viruses, which are able to infect more than 80 different host species. They are a diverse group with over 400 types discovered, of which almost half infect humans. Human papillomaviruses have been linked to a myriad of diseases, including multiple cancers, recurrent respiratory papillomatosis, and genital warts. The disease burden of HPV-related conditions is severe, and there is currently no treatment that can guarantee eradication of viral infection. All PV types characterised so far have a similar genomic structure, and contain the so called 'core ORFs' – E1, E2, L1 and L2 which are essential for viral genome replication and packaging into infectious virions. PV evolution and diversification appears to have been impacted by the availability of certain epithelial niches, with co-evolution and niche adaptation allowing PVs to develop a remarkable species and tissue specificity. Consequently, the function of the PV early proteins can vary between different PV species and types, but as a group they share important organisational similarities that reflect their common requirement to infect and persist in the epithelium following infection. This has allowed the use of animal models to gain insight into the basic virus/host interactions that are targeted by this group of viruses as a whole. The mechanisms by which HPV establishes a lesion, particularly in low-risk types, are not fully understood. However, the recently identified mouse model of PV infection is a useful biological tool to study this period of PV infection in vivo.

This body of work aims to expand current knowledge of early events in the PV life cycle. To further understand the mechanisms of cell persistence during PV infection, immunodeficient mouse tail samples inoculated with MmuPV1 were examined to investigate early lesion formation. Five discrete stages of lesion formation were characterised in the immunodeficient animals. In parallel studies, microlesions were rarely observed in immunocompetent C57BL/6J mice, reaching stage three of lesion formation. In-depth tissue analysis suggested a modulation of basal cell density in infected epithelium, and a delay in normal differentiation commitment in E6/E7 expressing cells. Whole genome cell culture experiments were attempted in parallel with human high-risk types, which showed a post-confluent effect of high concentration EGF on cell growth and genome copy number in cells containing HPV16 genomes. A role for MmuPV1 E6 in growth of cell populations to significantly higher densities was shown through experimentation with cells exogenously expressing viral proteins. Differentiation was also delayed in the cells expressing MmuPV1 E6, demonstrating a recapitulation of events characterised in in vivo infections. Novel use of fluorescent cell lines in tandem with confocal microscopy allowed innovative analysis of a high-density monolayer cell culture model. These experiments revealed that MmuPV1 E6 expression resulted in preferential persistence of cells in the lower layer over cells expressing control vector only. Disruption of MmuPV1 E6 binding with MAML1 protein abrogated this phenotype, suggesting that this interaction was necessary for the lower layer persistence phenotype shown by MmuPV1 E6 expressing cells.

Overall, the findings of this thesis suggest that expression of MmuPV1 E6 confers a competitive advantage on infected cells in the basal layer of the epithelium, allowing expansion of the reservoir of infection. Patterns of virus gene expression suggest a related but distinct life cycle phenotype for MmuPV1, a pi papillomavirus type, when compared to alpha papillomaviruses. Wherein, amplification begins immediately upon basal layer exit as opposed to the exit and reentry phenotype suggested in high-risk lesions. Further characterisation of these phenotypes will likely provide important information on key mechanisms in early lesion formation, and it is reasonable to consider that pi papillomaviruses may serve as a better model for beta papillomaviruses than alpha types.





Doorbar, John


virology, papillomavirus, human papillomavirus, mouse model, homeostasis, cell biology, keratinocyte


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge