Diversity in Papillomavirus Host Interactions During Attachment and Entry
Open Access
- Author:
- Cruz, Linda Marie
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 25, 2013
- 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
Clare E Sample, Committee Member
Janette Marie Mcallister, Committee Member - Keywords:
- Human papillomavirus
HPV
Viral Entry
Virus Attachment
Cell Binding
Glycosaminoglycans
Furin - Abstract:
- Infections by high-risk human papillomaviruses (HPV) are the main causative agents for the development of cervical cancer. HPVs are uniquely associated with and dependent on host epithelial stratification and differentiation for completion of the viral life cycle. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV) many host cellular proteins have been implicated in the process. Data reported in literature suggest there are structural and functional differences between recombinant and tissue-derived HPV particles. PsV particles are produced by over-expression of the viral capsid proteins in monolayer cells and thus bypass the need for stratified and differentiated epithelial tissue. Knowledge of attachment and entry by tissue-derived HPV particles is lacking due to the more efficient production and ease of use of PsV particles. The objective of this thesis was to investigate some of the cell surface interactions reported to play a role in attachment and entry, using tissue-derived HPV particles. Further, we wanted to explore whether some of the most common cancer-causing HPV types shared the same requirements. Glycosaminoglycans (GAGs), especially heparan sulfate (HS), are the primary attachment receptor mediating infection by HPV PsV. Here we show that native HPV particles produced in a differentiated epithelium have developed different strategies to infect the host. In biochemical inhibition assays using purified GAG polysaccharides and GAG-negative cell, the four most common cancer-causing HPV types, HPV18, HPV31, and HPV45 were largely dependent on GAGs to initiate infection. However, their specificities varied. In contrast, HPV16 could bind and enter through a GAG-independent mechanism. Infections of primary human keratinocytes, natural host cells for HPV infections, supported our conclusions. The proprotein convertase furin cleaves the N-terminus of the minor capsid protein L2 post-attachment to host cells and is required for infectious entry by HPV PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that infecting with tissue-derived HPV16 native virus (NV) was independent of cellular furin. We show that HPV16 L2 may be cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 was also not dependent on cellular furin for infection, but infecting with two other alpha papillomaviruses, HPV18 and HPV31, were dependent on the activity of cellular furin. We extended our analysis of GAG- and furin-dependence to HPV16 molecular variants. HPV16 variants of non-European origin are associated with an increased risk for the development of cervical cancer. Our data suggests that HPV16 variants, similarly to different HPV types, have different specificities for interactions with cellular molecules for infection of the host. Importantly, current HPV vaccines target the major capsid protein L1 of HPV16 and HPV18, which together account for 70% of cervical cancer cases, protecting against virus infection and development of neoplasias. However, since current vaccines are type-specific, they do not offer protection against all cancer-causing HPV types. In addition, they are cost-prohibitive to most women around the world. Thus, there is a need for the development of less expensive alternatives, such as universal microbicides in addition to the current vaccines. We tested carrageenan, a polysaccharide that has microbicidal activities against HPV PsV particles by competing with virus-GAG interactions. Similarly to the different GAG-specificities of the various tissue-derived HPV types, they displayed differential susceptibilities to carrageenan. Our data demonstrate that ordered maturation of papillomavirus particles in a differentiating epithelium may alter the virus entry mechanism. This study should facilitate a better understanding of the attachment of and infection by the main oncogenic HPV types. Further, our work sets a framework f0or future research on attachment and entry by tissue-derived particles and for the development of inhibitors of HPV infection.