Advisory Committee Chair
Louise T Chow
Advisory Committee Members
Thomas R Broker
Weei-Chin Lin
J Michael Ruppert
Thomas M Ryan
Hengbin Wang
Document Type
Dissertation
Date of Award
2010
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
The human papillomavirus (HPV) establishes persistent infections in the basal stratum of squamous epithelia, while productive amplification of viral DNA occurs in differentiated keratinocytes prior to virion assembly in the superficial strata. Until recently, only in situ hybridization (ISH) of low-grade HPV lesions could be used to reveal a snap shot of the viral life cycle. There has been a critical need to reproducibly propagate HPV infections in culture for consistent genetic analyses. Organotypic raft cultures recapitulate a differentiated squamous epithelium. Our lab utilized in vivo Cre-mediated recombination to reconstitute the entire HPV-18 genome in primary human keratinocytes (PHKs). My fluorescent in situ hybridization (FISH) to examine HPV DNA and RNA revealed robust differentiation-dependent viral activities and enabled a redesign that ultimately generated abundant progeny virus. Crucially, harvested virus elicited a new cycle of the productive infectious program in PHKs. The contextual progression of raft cultures containing HPV-18 replicons was consistent with known patterns of viral DNA amplification. Importantly, a detailed view of virus-host interactions was achieved by simultaneous in situ probing for viral DNA, viral proteins and host cell biomarkers. S-phase reentry was induced in numerous differentiated suprabasal epithelial cells on days 8 and 10. Unexpectedly, the patterns of HPV DNA amplification did not parallel those of the stochastic S phase cells. Rather, I show that viral DNA amplification initiated from G2 arrested on days 10 and 12 in the spinous strata. These cells accumulated high levels of cytoplasmic cyclin B1. While ectopic expression of HPV E1^E4 leads to G2 arrest in cell lines, I demonstrate that this viral protein accumulated following cyclin B stabilization and coincidental with the high amplification of viral DNA. Interestingly, such differentiated cells with high copies of viral DNA lost HPV E7 activity and transitioned to express capsid proteins for virion morphogenesis. An immortalization-null HPV-18 E6*I mutant genome was deficient in viral DNA amplification and failed to express the major capsid protein. These observations emphasize the value of simultaneous multiplexed detections in discerning the subtle interplay between naturally progressing HPV pathogenesis within its differentiating environment.
Recommended Citation
Duffy, Aaron A., "Visualizing the Productive Program of HPV in Differentiating Squamous Epithelial Tissue" (2010). All ETDs from UAB. 1559.
https://digitalcommons.library.uab.edu/etd-collection/1559