title: Subcellular localization and topology of the p7 polypeptide of hepatitis C virus.
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.
authors.affiliation: CNRS-FRE2369, Institut de Biologie de Lille/Institut Pasteur de Lille, 59021 Lille Cedex, France.
authors.forename: Séverine
authors.initials: S
authors.lastname: Carrère-Kremer
authors.forename: Claire
authors.initials: C
authors.lastname: Montpellier-Pala
authors.forename: Laurence
authors.initials: L
authors.lastname: Cocquerel
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Wychowski
authors.forename: François
authors.initials: F
authors.lastname: Penin
authors.forename: Jean
authors.initials: J
authors.lastname: Dubuisson
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000086082:Viroporin Proteins
chemical_list: D008565:Membrane Proteins; D021382:Protein Sorting Signals; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins