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Journal of Virology, June 2007, p. 6682-6689, Vol. 81, No. 12
0022-538X/07/$08.00+0     doi:10.1128/JVI.02783-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Binding Dynamics of Hepatitis C Virus' NS5A Amphipathic Peptide to Cell and Model Membranes

Nam-Joon Cho,^1,3, Kwang Ho Cheong,^3,, ChoongHo Lee,³ Curtis W. Frank,^2* and Jeffrey S. Glenn^3,4*

Department of Materials Science and Engineering, Stanford University, Stanford, California 94305,¹ Department of Chemical Engineering, Stanford University, Stanford, California 94305,² Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford University, Stanford, California 94305,³ and Veterans Administration Medical Center, Palo Alto, California 94304⁴

Received 17 December 2006/ Accepted 7 March 2007

Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.

Published ahead of print on 11 April 2007.

These authors contributed equally.

Present address: Bio Lab, Samsung Advanced Institute of Technology, Yongin-Si, Gyeonggi-Do, Korea 449-712.