Human papillomaviruses (HPVs) infect mucoasal and cutaneous epithelium. About 6.2M new cases of sexually transmitted HPV infections are reported every year, and >20M people in the US are currently infected. HPV infections cause anogential cancers, especially cervical cancer, and are linked to >99% of all cervical malignancies. HPV types 16 and 31 cause anogenital infections and related cancers, but each causes a distinct infectious outcome and the viruses have different biological characteristics. Major obstacles in HPV research have included the lack of high-titer infectious viral stocks and the lack of an in vitro assay system for the study of early infection phases and viral gene expression. We have been successful in generating high-titer HPV virion stocks and we show evidence that oncogenic HPV types HPV16 and HPV31 interact differently with normal host cells, human keratinocytes (HKs). HPV16 has an internalization half time (t1/2) of ~4h, whereas the t1/2 for HPV31 is ~14h;HPV16 enters via clathrin-coated pits, but HPV31 uses a clathrin-independent, caveolae- and dynamin 2-dependent pathway in HKs that involves lipid rafts. Unlike previous studies, we will investigate bona fide HPV infections in normal host HKs. We will use a combination of multi-parameter confocal and video microscopic visualization (localization) and infectious entry assays (detection of viral transcription) to assess the routes of infection of normal host cells. Our central hypothesis is that specific external viral capsid features dictate the dissimilarities in the early infection events of HPV16 and HPV31 in HK. We will exploit the differences between HPV16 and HPV31 to pursue this program's goals, which are to identify the viral and cellular factors and define the mechanisms dictating the viral infection process in host HKs. To test our hypothesis, we will pursue three inter-related Specific Aims:
In Aim 1 we will determine the viral and cellular factors affecting initial interactions with HKs. This will employ mixed virions comprised of HPV16 L1+HPV31 L2 (or HPV31L1+HPV16L2) and video microscopy of single particle tracking on host cells.
In Aim 2 we will determine the requirement for intracellular signaling in virion entry and trafficking.
In Aim 3 we will use novel dual fluor-labeled infectious particles created in our lab to delineate the route of intracellular trafficking and the location of virion uncoating. These studies will reveal the intracellular vesicles involved, and investigate the role of L2 in vesicle escape. We are employing established techniques for studying virus internalization and trafficking, now possible for HPVs due to the availability of high-titer viral stocks. This work will provide a foundation for defining the molecular mechanisms that control the establishment of HPV infections. Understanding the processes of viral entry will impact future development of broadly cross-protective prophylactic strategies for preventing persistent oncogenic HPV infections, a major risk for morbidity and malignant progression. Our findings will also provide significant insight into receptor-mediated endocytosis in keratinocytes, an area vastly understudied.

Public Health Relevance

Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the US, and cause a variety of benign and malignant neoplasia of the skin and mucosal surfaces. This includes all cervical cancers, many other anogenital warts and cancers, H25% of head-and-neck cancers, and some skin cancers. Although vaccines are available for two to four HPV types (of over 100 total types), vaccination does not prevent infection by all HPVs, and numerous women and most men throughout the world, especially in developing countries, will remain unvaccinated. Currently e20 million Americans have sexually-transmitted HPV infections, with 6.2M new cases reporter each year. Our study is aimed at understanding the mechanisms how these viruses interact with and enter host cells to initiate infection, which may allow the design of more broadly acting agents capable of inhibiting infections by a wider array of HPV types.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA132136-04
Application #
8197232
Study Section
Virology - A Study Section (VIRA)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
2008-12-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
4
Fiscal Year
2012
Total Cost
$301,913
Indirect Cost
$100,638
Name
University of New Mexico
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
868853094
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Surviladze, Zurab; Sterkand, Rosa T; Ozbun, Michelle A (2015) Interaction of human papillomavirus type 16 particles with heparan sulfate and syndecan-1 molecules in the keratinocyte extracellular matrix plays an active role in infection. J Gen Virol 96:2232-41
Ozbun, Michelle A; Patterson, Nicole A (2014) Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses. Curr Protoc Microbiol 34:14B.3.1-14B.3.18
Surviladze, Zurab; Sterk, Rosa T; DeHaro, Sergio A et al. (2013) Cellular entry of human papillomavirus type 16 involves activation of the phosphatidylinositol 3-kinase/Akt/mTOR pathway and inhibition of autophagy. J Virol 87:2508-17
Dziduszko, Agnieszka; Ozbun, Michelle A (2013) Annexin A2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in human keratinocytes. J Virol 87:7502-15
Bergant MaruĊĦi?, Martina; Ozbun, Michelle A; Campos, Samuel K et al. (2012) Human papillomavirus L2 facilitates viral escape from late endosomes via sorting nexin 17. Traffic 13:455-67
Campos, Samuel K; Chapman, Janice A; Deymier, Martin J et al. (2012) Opposing effects of bacitracin on human papillomavirus type 16 infection: enhancement of binding and entry and inhibition of endosomal penetration. J Virol 86:4169-81
Surviladze, Zurab; Dziduszko, Agnieszka; Ozbun, Michelle A (2012) Essential roles for soluble virion-associated heparan sulfonated proteoglycans and growth factors in human papillomavirus infections. PLoS Pathog 8:e1002519
Campos, Samuel K; Ozbun, Michelle A (2009) Two highly conserved cysteine residues in HPV16 L2 form an intramolecular disulfide bond and are critical for infectivity in human keratinocytes. PLoS One 4:e4463