Investigating the role of the E5 viroporin of human papillomavirus (HPV) in transformation and the virus life cycle

Cervical cancer is a leading cause of death worldwide and is almost universally associated with infection with the human papillomavirus (HPV). In addition, HPV infection is also associated with an increasing number of other cancer that can affect both sexes; these include cancer of the head and neck. Not all people who are infected with HPV will develop cancer but it is thought that some of the protein building blocks produced by the virus are responsible for changing the nature of the cells that they infect so that they may eventually develop into cancerous cells. HPV expresses three of these "oncoproteins". Out of the three, the E5 protein is the least understood. This is probably due to the difficult nature of this protein that makes it hard to study.
We have recently shown that E5 is able to form ring-like structures that act as a channel with a central pore that forms holes in artificial membranes that we can use to mimic the inside of cells. It is thought that this pore must allow small molecules and ions to pass through it, and perhaps this causes changes within the environment of the infected cell. We used computer based simulations of the channel to design small molecules that could block this pore and we found that these inhibitors were also able to prevent some of E5's known effects in cells.
We now want to take this research further to try and understand how this channel works, what makes it open or close and can we build better inhibitors to switch it off. In addition, we want to know what the channel does in cells and whether the virus needs this channel for its life cycle. For these studies we will use cell culture experiments in the laboratory and focus on some well know effects of expressing E5 in cells.
These studies should give us a clear picture of whether the E5 channel is essential for the HPV life cycle. If so then this opens up the possibility of targeting this protein for anti-viral therapeutics.

Human papillomavirus (HPV) is a major human pathogen and the major cause of cervical cancer and a growing number of orogenital malignancies in both sexes. Despite the recent development of the vaccines, the long incubation period of the virus and the ineffective nature of the vaccine in HPV-exposed individuals will hinder reductions in HPV-associated cancers. This is a significant health burden, given that HPV screening and treatment costs the USA alone $3 billion per annum. Development of effective treatments to compliment the vaccine strategy is clearly warranted.
To develop directly acting antivirals, a detailed understanding of HPV biology is required. The E5, oncoprotein has remained poorly studied. E5 expression causes tumours in the skin/cervix of mice and this is thought to occur by a mechanism that required deacidification of endosomes to deregulate EGFR signalling in cells. how this is brought about is the subject of considerable controversy.
We demonstrated that E5 is a member of the viroporin family of channel forming proteins. Furthermore, using in silico molecular modelling techniques we generated small molecule inhibitors of the E5 channel. These reduced EGFR hyperactivation in cell culture, providing the first evidence of a viroporin with transforming abilities.
We will test the hypothesis that the E5 viroporin directly alkalinises EGFR containing endosomes to deregulate EGFR trafficking. We will combine our liposome technologies with in silico molecular modelling to understand channel function and inform cell based assays to study EGFR signalling. We will perform a comprehensive analysis of the role of the E5 viroporin in the HPV life cycle studies, we using organotypic raft cultures. These are the gold standard system to study the HPV life cycle. This proposal seeks to significantly increase our understanding of this poorly characterised oncoprotein, with the long-term aim of validating E5 as a possible target for antiviral intervention.

Anogenital and oropharngeal cancers caused by human papillomavirus are important medical problems worldwide. Whilst the development of HPV vaccines is a great stride forwards, they are raised against a limited number of HPV types and are only protective in immunocompetent and HPV-naïve individuals, so will not protect the many millions already infected with HPV. Social and economic challenges make widespread administration of the vaccine difficult, particularly in developing countries. In conjunction, virological issues including the long latency period of infection make it likely that significant decreases in HPV-associated cancers will be slow to manifest. As a result, there remains a large cohort of untreated individuals infected with a virus that may give rise to cancer. Indeed, patients harbouring HPV16 are at approximately 40% risk of developing premalignant cervical intraepithelial neoplasia after 3-4 years infection. This results in the annual diagnosis of approximately 400,000 new cases of CIN in the USA alone, with annual costs of $2.3 billion for cervical cancer screening and $700 million for treatment.
Such statistics have prompted intensive research into the development of therapies that directly target HPV infected cells, which might act prior to the development of invasive cancer, to compliment rather than compete with the vaccines. This grant proposal is rooted in basic science, and primary beneficiaries in the short term will be the UK and worldwide academic community of virologists and cell biologists, who will be able to build on our data studying HPV and other cancer causing viruses. We anticipate this impact will first occur within one year of the proposal, when our initial results are presented at national and international meetings and published in appropriate journals. We anticipate high productivity from this grant, and so impact in this manner will continue throughout the course of the funding period.
In addition to academic colleagues, we envisage that the commercial sector may wish to utilise our findings to develop small molecule inhibitors against E5 as part of a potential novel chemotherapeutic strategy. Ultimately, this will enhance the economic competitiveness of the UK, improve the skills base of UK-based scientists and ultimately may provide part of an effective anti-HPV chemotherapeutic strategy. To facilitate this transition from the basic science proposed in this application to translational research, the University of Leeds has several mechanisms already in place, including seed funding, dedicated personnel with industrial and knowledge transfer expertise, and previously established commercial/academic partnerships. The ways in which we will exploit these resources is detailed in the 'pathways to impact' document that accompanies this proposal.
The final group of beneficiaries is the public. At the UoL and Astbury Centre for Structural Molecular Biology we have strong links with local schools and museums, particularly the Thackray Museum (The Museum of Modern Medicine, Leeds). We regularly work with the museum on biologically and medically relevant science being undertaken at UoL. In addition, members of FBS (including Macdonald) routinely visit local secondary schools to explain the purpose of their research. In this way the public will be made aware of the efforts of academic institutions to safe guard the nation's health.