Evidence for the Formation of a Heptameric Ion Channel Complex by the Hepatitis C Virus P7 Protein in Vitro

• Published in: The Journal of biological chemistry 2006-12, Vol.281 (48), p.37057-37068
• Main Authors: Clarke, Dean, Griffin, Stephen, Beales, Lucy, Gelais, Corine St, Burgess, Stan, Harris, Mark, Rowlands, David
• Format: Article
• Language: English
• Subjects: Amantadine - chemistry; Animals; Chromatography, High Pressure Liquid; Drug Design; Electrophoresis, Polyacrylamide Gel; Escherichia coli - metabolism; Glutathione Transferase - metabolism; Hepatitis C virus; Ion Channels - chemistry; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Microscopy, Electron, Transmission; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Swine; Viral Proteins - chemistry; Viral Proteins - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M602434200

The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.