A Study of Native Human Papillomavirus Structure and Capsid Stabilty
Open Access
- Author:
- Ryndock, Eric Joseph
- Graduate Program:
- Microbiology and Immunology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 05, 2015
- Committee Members:
- Craig Matthew Meyers, Dissertation Advisor/Co-Advisor
- Keywords:
- HPV
Virus
Capsid
Stability - Abstract:
- Human papillomavirus (HPV) is the most common sexually transmitted virus and infects through skin to skin contact. This virus is the main causative agent of cervical cancer and remains a major human health problem. HPV capsids are composed of the major capsid protein, L1, and the minor capsid protein, L2. Viral capsids are held together through a complex network of hydrophobic interactions and disulfide bonds. Historically, studying HPV was difficult, as viral production was limited to expensive animal models. Eventually, recombinant systems using monolayer cells transfected with L1 expression vectors were created to produce viral particles. These particles retain properties of native virions, but are produced in the absence of cellular differentiation. This method led to the generation of a first phase HPV vaccine to prevent HPV infection. The similarities of recombinant virions and virions produced in a differentiated epithelium are not understood. Organotypic raft technology is an alternative to recombinant systems that produces HPV in an in vitro, differentiated epithelium. The goal of this dissertation was to study HPV capsid structure and stability of native virions produced in the organotypic raft system. From this study, vulnerabilities in HPV capsid structure could be exploited to design future vaccines or disinfectants against the virus. Specifically, we investigated the biological significance of methionines in-frame of the L1 open reading frame (ORF) and upstream of the predicted translational start site of L1, conserved cysteines in the L1 major capsid protein thought to mitigate capsid assembly, and the efficacy of disinfecting HPV using clinical disinfectants. We explored the biological significance of upstream methionines found in the L1 ORF in two of the most common HPV types that to cause cancer in humans, HPV16 and HPV18. Recombinant systems use expression vectors containing non-native HPV codons and promoters to produce L1. These expression vectors are designed to produce L1 starting from a consensus methionine found by aligning the N-terminus of the L1 ORF from different HPV types. In many HPV types, the consensus methionine is not the first in-frame methionine in the L1 ORF [11]. HPV18 has two upstream, in-frame methionines, which are 61 (M(-61)) and 26 (M(-26)) amino acids away from the consensus methionine (M(1)). We found that HPV18 produce two different L1 isoforms that originate from M(-61) and M(1). Both forms of L1 are included in the virion. Silencing either M(-61) or M(1) caused changes in virus stability, infectivity, and conformation. L1 cysteines are important to providing stabilizing disulfide bonds in the final conformation of the capsid. C175, C185, and C428 are the cysteines responsible for forming disulfide bonds between L1 pentamers in HPV16. It was reported that HPV16 VLPs containing mutations in C161, C229, and C379 have less stable capsids than wild-type. These cysteines do not participate in disulfide bonds linking pentamers in the mature capsid. We hypothesized that if C161, C229, and C379 are involved in the final structure of the capsid, it occurs during the early events of capsid assembly. We show that these cysteines contribute to the final capsid conformation, potentially through the formation of transitional disulfide bonds. The development of an HPV vaccine has provided protection against HPV infection and in extension, cervical cancer. However, HPV DNA has been detected on human finger tips. Free papillomavirus genomes cause papillomas in animal models. Also, virions are protected inside sloughed off keratinocytes, making them an excellent candidate for long-term survival on surfaces. All of these factors lead to the potential of HPV being transmitted through non-sexual avenues, such as fomites. HPV disinfection guidelines are derived from similar viruses. There are no studies that utilize native virions. We found that a majority of the disinfectants used in healthcare settings are ineffective at neutralizing HPV. These data in this thesis demonstrate multiple mechanisms in which HPV structure and stability are controlled in differentiated epithelium and how current disinfectants fair against destroying the virus. This study should expand the knowledge about HPV structure and capsid stability in its natural environment, as well as aid in the design of future vaccines and disinfectants to prevent infection.