Genetic and Biochemical Analyses of Human Papillomavirus Capsid Assembly and Maturation
Open Access
- Author:
- Conway, Michael James
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 01, 2010
- Committee Members:
- Craig Matthew Meyers, Dissertation Advisor/Co-Advisor
Craig Matthew Meyers, Committee Chair/Co-Chair
Neil David Christensen, Committee Member
David Joseph Spector, Committee Member
Jianming Hu, Committee Member
John Michael Flanagan Jr., Committee Member - Keywords:
- Differentiation
Redox
Maturation
Assembly
Capsid
Human Papillomavirus
Disulfide bond - Abstract:
- High-risk human papillomavirus (HPV) types are the etiological agents of numerous malignant neoplasias of the anogenital and oropharyngeal mucosae. Our understanding of native HPV (NV) capsid assembly and maturation events within its natural host environment- stratifying and differentiating human epithelial tissue is lacking due to the widespread utilization of recombinant HPV particles. Recombinant particles bypass the need for stratified and differentiated tissue since they are produced via the overexpression of capsid proteins in monolayer culture. The objective of this thesis was to investigate the mechanisms of NV assembly and maturation within the context of its natural host environment. Included within this thesis are novel qPCR-based assays that allow for the specific infectivity of wild-type and mutant NV to be assessed, in addition to novel techniques to assess the relative stabilities of wild-type and mutant capsids. Recent research suggests that recombinant, HPV pseudovirions (PsV), which are produced in monolayer culture, can undergo a redox-dependent conformational change that takes place over the course of many hours. This conformational change is dependent on the formation of disulfide bonds. It is also characterized by resistance to proteolysis and chemical reduction, and the appearance of more orderly capsid structures via transmission electron microscopy (TEM). In order to understand the kinetics and mechanisms of NV assembly and maturation within stratifying and differentiating epithelial tissue, electron microscopic analysis in addition to immunohistochemical staining were performed on HPV16-infected 10, 15, and 20-day organotypic raft tissues. We found a temporal transition of NV capsids from the chemically reducing suprabasal nuclei within 10-day-old tissue to the chemically oxidizing cornified envelope within 15 and 20-day-old tissue. Importantly, we showed that stability and specific infectivity of NV extracted from 20-day-old tissue increased over that of NV extracted from 10-day-old tissue. Also, both viral DNA encapsidation and infectivity of HPV-infected tissues were redox-dependent in that they were positively manipulated via the treatment of organotypic tissues with oxidizing reagents. These effects were concentration and temporally-dependent. Conversely, both viral DNA encapsidation and infectivity of HPV-infected tissues were negatively manipulated via the treatment of organotypic tissues with the organic and inorganic antioxidants which supported a role for a tissue-spanning redox gradient in the production and stabilization of HPV capsids. Further, the redox-dependent enhancement of capsid stability and genome encapsidation was altered in HPV16 NV containing serine substitutions for the conserved L1 cysteine residues, C175, C185, and C428. We propose from these studies that a regulated assembly process occurs within stratifying and differentiating epithelial tissue whereby C185-C428 disulfide bonds form first, followed by final C175-C428 interpentameric disulfide bonds within mature, 20-day NV. Exposure of HPV16 L2 cross-neutralizing epitopes also appeared to be temporally regulated through a redox-dependent mechanism, with 20-day native HPV16 virions being more susceptible to neutralization than 10-day virions. However, this temporal susceptibility to neutralization did not extend to native HPV31 or HPV18 virions, since they were equally susceptible to cross-neutralization at both 10 and 20-days. Further, substitution of the N-terminal conserved L2 cysteine residues, C22 and C28, for serine showed overlapping yet distinct functional roles for these residues. Substitution of either residue led to less effective neutralization of native HPV16 virions; however, C22S mutations led to a more dramatic resistance to neutralization. Conversely, whereas the C28S substitution greatly reduced the stability of 20-day capsids, 20-day native C22S virions remained as stable as wild-type. As a result of work in this thesis, we hypothesize that, in the context of stratifying and differentiating human epithelial tissue, HPV may take advantage of a tissue-spanning redox gradient in order to form critical disulfide bonds necessary for assembly and maturation. We propose that initial capsid assembly occurs within the reducing environment of suprabasal nuclei through mostly hydrophobic interactions. These hydrophobic interactions are then reinforced by disulfide bonds as the virions are pushed up through the epithelial strata, into the oxidizing environment of the cornified envelope. Our studies highlight the synergy between utilizing recombinant and native papillomavirus particles to uncover novel aspects of the HPV life cycle, and provides a framework for the continued study of HPV assembly within its natural host environment.