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.
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.
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.
