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

Author:

Carrère-Kremer Séverine1,Montpellier-Pala Claire1,Cocquerel Laurence1,Wychowski Czeslaw1,Penin François2,Dubuisson Jean1

Affiliation:

1. CNRS-FRE2369, Institut de Biologie de Lille/Institut Pasteur de Lille, 59021 Lille Cedex

2. CNRS-UMR5086, Institut de Biologie et Chimie des Protéines, 69367 Lyon Cedex 07, France

Abstract

ABSTRACT 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.

Publisher

American Society for Microbiology

Subject

Virology,Insect Science,Immunology,Microbiology

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