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Journal of Virology, April 2002, p. 3720-3730, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3720-3730.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

Séverine Carrère-Kremer,1 Claire Montpellier-Pala,1 Laurence Cocquerel,1,{dagger} Czeslaw Wychowski,1 François Penin,2 and Jean Dubuisson1*

CNRS-FRE2369, Institut de Biologie de Lille/Institut Pasteur de Lille, 59021 Lille Cedex,1 CNRS-UMR5086, Institut de Biologie et Chimie des Protéines, 69367 Lyon Cedex 07, France2

Received 29 October 2001/ Accepted 16 January 2002

Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.

* Corresponding author. Mailing address: Unité Hépatite C, CNRS-FRE2369, Institut de Biologie de Lille, 1 rue Calmette, BP447, 59021 Lille Cedex, France. Phone: (33) 3 20 87 11 60. Fax: (33) 3 20 87 11 11. E-mail: jean.dubuisson{at}ibl.fr.

{dagger} Present address: Stanford University School of Medicine, Stanford, CA 94305-5151.

Journal of Virology, April 2002, p. 3720-3730, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3720-3730.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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