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.
Hepatitis C virus (HCV)5 currently infects over 3% of the world's population and is the major indicator for liver transplantation in the developed world. Liver disease in the form of cirrhosis, liver failure, or hepatocellular carcinoma arises after many years of persistent virus infection following sub-clinical acute episodes. For this reason intervention is limited to chemotherapeutic treatment of chronic patients. The current regime of pegylated interferon ␣ and ribavirin, while effective against certain viral genotypes, is largely ineffective against the most common HCV genotype 1 (1, 2) viruses making the search for new HCV anti-viral drug targets crucial. A recent metaanalysis of multiple clinical trials where amantadine was included alongside current therapies showed that an improved sustained viral response was achieved in patients that had previously failed to respond to dual therapy (3).HCV is the prototype member of the Hepacivirus genus of the Flaviviridae family. It is an enveloped virus, and its genome comprises a single-stranded positive sense RNA of ϳ9.6 kb (4) that is translated from an internal ribosome entry site to yield a single polyprotein. This is cleaved by both host signalases and virus-encoded proteases yielding viral structural proteins and the non-structural proteins (5); the latter form the viral RNA replication complexes on modified cellular membranes and modulate host cell metabolism (6).The region of the HCV genome between the structural and non-structural regions encodes a 63-amino acid integral membrane protein known as p7 (7), comprising two trans-membrane domains separated by a short basic loop (7, 8). We identified the function of p7 as a cation channel whose activity in suspended lipid bilayers was sensitive to the drug amantadine, providing a potential mechanism for the proposed efficacy of this drug in HCV treatment (9). Others have subsequently identified alt...