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

Dean Clarke,Stephen Griffin,Lucy Beales,Corine St Gelais,Stan Burgess,Mark Harris,David Rowlands

doi:10.1074/jbc.m602434200

Abstract

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.

Highlights

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]
We previously described a bacterial expression system for p7 based on a glutathione S-transferase (GST) fusion methodology
As the 22-kDa band was reactive to the anti-FLAG antibody in Western blots and not to the antiGST antibody, it was thought to represent an N-terminal truncation of GST-FLAG-p7

Summary

Introduction

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]. We have used rotational averaging and harmonic analyses of TEM images to determine the precise stoichiometry of channel complexes formed by GST-p7 fusion proteins in a lipidic environment.

Results

Conclusion