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J. Biol. Chem., Vol. 281, Issue 48, 37057-37068, December 1, 2006

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

From the Institute of Molecular and Cellular Biology, University of Leeds, Faculty of Biological Sciences, Garstang Building, Leeds, West Yorkshire LS2 9JT, United Kingdom

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.

Received for publication, March 15, 2006 , and in revised form, October 5, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a studentship from the Medical Research Council.

² Supported by the Wellcome Trust (Grant 074023).

³ Recipient of a studentship from the Biotechnology and Biological Sciences Research Council.

⁴ To whom correspondence should be addressed: Tel.: 44-113-343-5641; Fax: 44-113-343-5638; E-mail: d.j.rowlands{at}leeds.ac.uk.

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