Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus p7 protein in vitro.
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.
Clarke
Dean
D
Institute of Molecular and Cellular Biology, University of Leeds, Faculty of Biological Sciences, Garstang Building, Leeds, West Yorkshire LS2 9JT, United Kingdom.
Griffin
Stephen
S
Beales
Lucy
L
Gelais
Corine St
CS
Burgess
Stan
S
Harris
Mark
M
Rowlands
David
D
D000547:Amantadine / Q000737:chemistry; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013552:Swine; D014764:Viral Proteins / Q000737:chemistry / Q000502:physiology*; D000086082:Viroporin Proteins
D000547:Amantadine; D005982:Glutathione Transferase; D007473:Ion Channels; D008051:Lipid Bilayers; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins