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Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system
Corine StGelais1, Tobias J Tuthill, Dean S Clarke
1Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Abstract:
Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target.
Insights
Developing new hepatitis C virus (HCV) therapies is crucial as current chemotherapy is often ineffective. Researchers created a novel assay to screen for drugs targeting the essential HCV p7 protein channel.
Area of Science:
- Virology
- Biochemistry
- Drug Discovery
Background:
- Hepatitis C virus (HCV) infection poses a significant global health challenge, with over 50% of patients unresponsive to current chemotherapy.
- The HCV p7 protein is vital for viral replication and exhibits membrane channel activity, presenting a promising target for novel antiviral therapies.
- Existing methods for characterizing p7 protein function are not suitable for high-throughput drug screening, necessitating the development of new assays.
Purpose of the Study:
- To develop a novel, convenient in vitro assay for measuring the channel-forming activity of the HCV p7 protein.
- To validate this assay for screening compounds that inhibit p7 channel function as potential therapeutic agents against HCV.
Main Methods:
- A liposome-based assay was developed utilizing the release of a fluorescent indicator dye to quantify p7 protein channel activity.
- Recombinant p7 protein from genotype 1b HCV was incubated with liposomes containing the dye.
- The effect of pH and known inhibitors (e.g., amantadine) on dye release was assessed to validate the assay's specificity and reliability.
Main Results:
- The assay demonstrated that recombinant HCV p7 protein induces dose-dependent release of fluorescent dye from liposomes.
- This p7-mediated dye release was enhanced under acidic pH conditions.
- The observed activity was confirmed to be due to the formation of a size-selective pore, not non-specific liposome disruption, and was inhibited by amantadine and other compounds.
Conclusions:
- A novel and convenient in vitro assay has been established to measure the channel activity of the HCV p7 protein.
- This assay effectively validates p7 channel function and its inhibition by specific compounds, paving the way for its exploitation as a therapeutic target.
- The developed system offers a valuable tool for drug discovery efforts aimed at developing new, effective treatments for chronic hepatitis C virus infections.
