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Journal of Virology, June 2009, p. 6257-6268, Vol. 83, No. 12
0022-538X/09/$08.00+0     doi:10.1128/JVI.02663-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of a Novel Determinant for Membrane Association in Hepatitis C Virus Nonstructural Protein 4B{triangledown}

Jérôme Gouttenoire,1 Valérie Castet,2 Roland Montserret,3 Naveen Arora,2,{dagger} Vincent Raussens,4 Jean-Marie Ruysschaert,4 Eric Diesis,3 Hubert E. Blum,2 François Penin,3 and Darius Moradpour1,2*

Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, CH-1011 Lausanne, Switzerland,1 Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany,2 Institut de Biologie et Chimie des Protéines, UMR 5086, CNRS, Université de Lyon, IFR128 BioSciences Gerland-Lyon Sud, F-69367 Lyon, France,3 Center for Structural Biology and Bioinformatics, Laboratory for Structure and Function of Biological Membranes, Faculté des Sciences, Université Libre de Bruxelles, B-1050 Brussels, Belgium4

Received 27 December 2008/ Accepted 30 March 2009

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic {alpha}-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic {alpha}-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

* Corresponding author. Mailing address: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, BU44/07/2421, Rue du Bugnon 44, CH-1011 Lausanne, Switzerland. Phone: 41 21 314 47 23. Fax: 41 21 314 47 18. E-mail: Darius.Moradpour{at}chuv.ch

{triangledown} Published ahead of print on 8 April 2009.

{dagger} Present address: Institute of Genomics and Integrative Biology, Delhi University, Delhi 110007, India.

Journal of Virology, June 2009, p. 6257-6268, Vol. 83, No. 12
0022-538X/09/$08.00+0     doi:10.1128/JVI.02663-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gouttenoire, J., Montserret, R., Kennel, A., Penin, F., Moradpour, D. (2009). An Amphipathic {alpha}-Helix at the C Terminus of Hepatitis C Virus Nonstructural Protein 4B Mediates Membrane Association. J. Virol. 83: 11378-11384 [Abstract] [Full Text]  


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