Pathogen-host omics analyses of human papillomavirus type 16 sub-lineages in a human epithelial organoid model

Pathogens such as human papillomaviruses (HPVs) have co-evolved with their hosts and form a molecular basis for common diseases. Persistent infection with the “high-risk” HPV type 16 (HPV16) is a potent cause of anogenital and oropharyngeal cancers. Taxonomic HPV16 sub-lineages, based on geographic origin of discovery, are noteworthy due to their variable tumourigenicity. In this dissertation, I present basic research and the resulting biotechnologies we developed, improved, and utilized to study their fascinating pathogen-host relationship with human stratified epithelia. A small number of variations in the E6 gene of HPV16, found in the D2 and D3 sub-lineages, lead to increased tumourigenic risk compared to the prototype A1 sub-lineage. Using an organotypic human epithelial model (or in vitro organoid) we recapitulated the viral life cycle and used “-omics” analyses to assess viral and host molecular differences due to sub-lineage variation. Sub-lineage variants of E6 were associated with host genome instability and viral integration into host DNA. Following these initial findings, I provide perspective on epithelial organoids, namely that the trade-off between model complexity and feasibility should be sensibly considered based on its utility for answering the biological research question at hand. Model applications and improvements are presented, including time-series epithelial stratification measurements, strategies for introducing full-length sub-lineage HPV16 genomes into host keratinocytes, and experiments to study innate immune evasion. These wet-lab works are accompanied by software to aid biologists in analyzing sequencing data. As well, we present current work using The Cancer Genome Atlas to test the association between HPV16 sub-lineage and integration. Overall, this interdisciplinary and interconnected collection has significance for basic researchers, providing insight on how a small number of natural viral variations can lead to increased tumourigenic risk, as well as for experimentalists to gain insight on organoid modelling and novel bioinformatics tools. More broadly, characterizing these molecular interactions between pathogen and host enables us to form a basis for diagnosis, treatment, and ultimately prevention of disease. Future research should aim to closely integrate biological and computational sciences for improving experimental approaches and our ability to make meaningful biological interpretations given the complexity and variability of biological systems

Pull-down of human papillomavirus 16 E6 variants for interactome analysis: a methods development and analysis

Human Papillomavirus 16 (HPV16) is a double-stranded DNA virus known as a causative agent in almost all cervical cancers and an increasing number of oropharyngeal cancers. Variants of HPV16, such as the Asian-American (AA) and L83V, have been found to have increased abilities to promote carcinogenesis. Even though previous interactome studies identified which proteins interact with HPV16 E6, few have looked at interactions between variants of E6 in particular AAE6 and the European Prototype (EPE6). This thesis had two objectives: develop a method to co-immunoprecipitate host cellular proteins that interact with E6 variants; and identify potential differences between cellular host proteins and variant E6. We were successful in developing a method that not only could pull-down and identify E6 variant interacting proteins but the E6 variant proteins themselves. Using liquid chromatography-mass spectrometry (LC MS/MS), we identified 13 proteins that interact with both AAE6 and EPE6, along with six unique AAE6 interacting proteins and six unique EPE6 interacting proteins. Of the interactors we found, seven were of particular interest: TRIP12, GNL2, INO80B, CHMP4B, MX2, RPSK6K4A, and PROK2. These proteins affect a variety of cellular functions, including DNA replication and repair, telomere maintenance, cellular proliferation, ERK1/2 signaling, signal transduction, and immune response. Identification of different proteins using different bioinformatic analyses further provide evidence that AAE6 and EPE6 may have unique interactions with their host cells resulting in varied abilities to promote carcinogenesis. The identification of these proteins has furthered our understanding of potential mechanisms that allow AAE6 to promote carcinogenesis more than EPE6. More wet lab work is still required to confirm these interactions and determine their exact effects on the host cell