Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex

doi:10.1073/pnas.0807298105

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex

^†Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany;
^§Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland; and
^¶Institut de Biologie et Chimie des Protéines, UMR 5086, Centre National de la Recherche Scientifique, Université de Lyon, IFR 128 BioSciences Gerland-Lyon Sud, F-69397 Lyon, France

Communicated by Charles M. Rice, The Rockefeller University, New York, NY, July 29, 2008
↵^‡V.B., J.M.B., and R.M. contributed equally to this work. (received for review April 6, 2008)

Abstract

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane α-helix that may be involved in intramembrane protein–protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix α₀, formed by NS3 residues 12–23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

Footnotes

^‖To whom correspondence may be addressed. E-mail: f.penin{at}ibcp.fr or darius.moradpour{at}chuv.ch
Author contributions: V.B., J.M.B., R.M., F.P., and D.M. designed research; V.B., J.M.B., R.M., F.P., and D.M. performed research; V.B., J.M.B., R.M., H.E.B., F.P., and D.M. analyzed data; and H.E.B., F.P., and D.M. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The atomic coordinates of have been deposited in the BioMagResBank (BMRB) (accession nos. 15580 (NS4A[1-22]*) and 15582 (NS3[10-24]).
This article contains supporting information online at www.pnas.org/cgi/content/full/0807298105/DCSupplemental.