Interplay between hepatitis C virus structural proteins and the p7 ion channel during particle assembly

Lead Research Organisation: University of Leeds
Department Name: Inst of Molecular & Cellular Biology

Abstract

Over 170 million people currently live with hepatitis C virus (HCV); although most are not aware that they are positive as the initial illness is extremely mild. Decades later, however, symptoms appear and many patients require liver transplants due to the development of severe liver damage, known as cirrhosis, or even liver cancer. In developed countries, drug combinations can be used to treat these patients, although these only work in about 50% of cases and have severe side-effects. As a result of failed treatment, 10 000 individuals died from HCV infection last year in the USA alone.
Until very recently, researchers have been unable to grow HCV in the laboratory, which has severely limited progress on developing new drugs to combat infection. In 2005, however, a new strain of the virus that could be grown in culture was discovered. This meant that for the first time, the way in which individual virus particles are made within the cell could be studied, providing a new area for drug discovery.
Virus particles are made of a protein shell, or core, that surrounds the genome and many, including HCV, have a second membranous shell known as the envelope which contains proteins that allow the virus to enter new cells. The construction of a new particle within an infected cell is a complex process, so the virus hijacks host cell machinery to achieve it. One strategy the virus employs is to make proteins that alter the environment within the cell, making it suitable for the assembly of new virus particles. For HCV, this protein is known as p7. I discovered that p7 is able to form seven-membered pores in membranes, which alter how acidic various parts of the cell become. I also showed that a drug called amantadine can prevent p7 from doing this.
My study will test whether drugs like amantadine can be used to stop HCV spreading in culture, which is the first step towards developing new clinical treatments. I will then investigate how these pores affect the other particle components during assembly and where in the cell this takes place. I will then use a fluorescent HCV to track, under the microscope, the route which particles take when leaving a live cell. Lastly, I will determine which parts of the cellular machinery HCV manipulates in order to create new particles, which could highlight targets for future drug development.

Technical Summary

Hepatitis C virus (HCV) chronically infects over 3 % of the worlds!
population and is the leading indicator for liver transplant surgery. Unfortunately, the majority of carriers are unaware of their positive status as acute infection is usually asymptomatic, yet the majority ( 80 %) develop persistent infection. It is only after many years when symptoms present as a consequence of severe liver damage that clinical intervention takes place. Current therapy comprising interferon ?? combined with ribavirin, however, is expensive, poorly tolerated and ineffective in up to 50 % of cases. Thus, the search for new anti-HCV drugs is a priority, although these have been slow in development due to an inability to grow the virus in culture. Interestingly, clinical trials where amantadine was included alongside current therapies have shown encouraging results.
Recently, a cell culture system for HCV was described based on a unique virus isolate, JFH-1. This represents the most important break-through in HCV research to date and provides the first opportunity to study the processes involved in the formation of HCV virions. A key factor implicated in HCV assembly is the p7 protein, which I demonstrated to function as an amantadine-sensitive ion channel; thus providing a potential mechanism for the clinical action of the drug.
This proposal will build on my work characterising p7 and use JFH-1 to validate candidate p7 inhibitors as a means of blocking HCV replication in culture. Virus inhibition assays will be complemented by a novel in vitro assay for p7 channel function using recombinant protein. I will then use a unique anti-JFH-1 p7 antibody to examine the effects caused by a loss of p7 function on other HCV proteins, focusing on the stability of the viral core and envelope proteins. This will lead to an extensive examination of where HCV assembles within the cell and the route by which virions are secreted; combining live cell imaging of fluorescently labelled virus with cell fractionation, immunofluorescence and EM studies. The cellular processes hijacked by HCV to exit the cell will then be examined by characterising potential interactions with host vesiclular sorting machinery, providing further targets for therapy. Lastly, an additional role for p7 during virus entry will be investigated by determining whether p7 comprises part of the HCV virion. These studies will both provide an insight into a hitherto uncharacterised aspect of the HCV life-cycle and validate p7 as a target for future therapies.

Publications

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Griffin S (2010) Inhibition of HCV p7 as a therapeutic target. in Current opinion in investigational drugs (London, England : 2000)

 
Description Yorkshire Cancer Research Pump-priming grant
Amount £50,000 (GBP)
Organisation Yorkshire Cancer Research 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2011 
End 12/2011
 
Description royal society project grant
Amount £15,000 (GBP)
Funding ID RG081138 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2008 
End 04/2009
 
Description Chimeric GBVB/HCV with Dr Nicola Rose (NIBSC) 
Organisation National Institute for Biological Standards and Control (NIBSC)
Country United Kingdom 
Sector Public 
PI Contribution Leeds - Chimeras designed and generated and biochemical characterisation performed. GSK - animal work.
Collaborator Contribution NIBSC performed animal experiments on Tamarins infected with novel chimeric GBV-B expressing HCV p7 protein.
Impact Publication - 18845353
Start Year 2008
 
Description ESCRT pathway with Dr Colin Crump 
Organisation University of Cambridge
Department Department of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution Together identified the dependence of HCv on the cell ESCRT pathway during particle production.
Collaborator Contribution Provision of reagents and advice on research planning.
Impact Publication - 19828764 Oral presentations at Society for General Microbiology AGM 2008, UK HCV meeting 2009.
Start Year 2007
 
Description HCV assembly with Dr Chris McCormick 
Organisation University of Southampton
Department School of Medicine Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution Establishment of surrogate HCv assembly system
Collaborator Contribution Collaborative project on HCV assembly
Impact Publication - 19223490
Start Year 2008
 
Description Molecular Dynamics of p7 channels 
Organisation National Yang Ming University
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution We provided the atomic structure of p7 and the predicted binding site of novel inhibitors.
Collaborator Contribution Prof Wolfgang Fischer ran atomistic MD simulations of p7-inhibitor complexes, defining stability and key interactions.
Impact We are shortly due to submit a manuscript describing this work.
Start Year 2016
 
Description Novel GSk compounds 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution provision of reagents
Collaborator Contribution Provision of reagents
Impact Publication - 18828153
 
Description p7 function in cells with Prof Steven Weinman 
Organisation University of Kansas
Country United States 
Sector Academic/University 
PI Contribution Together investigated HCv p7 ion channel function in mammalian cells and correlated effect of antivirals with block of ion channel function
Collaborator Contribution Together investigated HCV p7 ion channel function in mammalian cells and correlated effect of antivirals with block of ion channel functionTogether investigated HCV p7 ion channel function in mammalian cells and correlated effect of antivirals with block of ion channel function
Impact International HCV meeting, poster presentation 2008, 2009. Oral presentation 2010. Publication 20824094.
Start Year 2007
 
Description p7 function in cells with Prof Steven Weinman 
Organisation University of Texas
Department University of Texas Medical Branch
Country United States 
Sector Academic/University 
PI Contribution Together investigated HCv p7 ion channel function in mammalian cells and correlated effect of antivirals with block of ion channel function
Collaborator Contribution Together investigated HCV p7 ion channel function in mammalian cells and correlated effect of antivirals with block of ion channel functionTogether investigated HCV p7 ion channel function in mammalian cells and correlated effect of antivirals with block of ion channel function
Impact International HCV meeting, poster presentation 2008, 2009. Oral presentation 2010. Publication 20824094.
Start Year 2007
 
Description Press release 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact Press release organised by University firm, "Campus PR"

Lead feature on University of Leeds Website, also posted in several HCV patient websites
Year(s) Of Engagement Activity 2008
 
Description Press release 2 - Hepatology paper 2011 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Press release taken up by local press, University, MRC and numerous patient websites as well as the Japanese Medical Tribune.

Numerous research groups have approached us to test their candidate p7 inhibitors.
Year(s) Of Engagement Activity 2011
URL http://www.leeds.ac.uk/news/article/2191/