Subcellular localization and topology of the p7 polypeptide of hepatitis C virus.
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.
Carrère-Kremer
Séverine
S
CNRS-FRE2369, Institut de Biologie de Lille/Institut Pasteur de Lille, 59021 Lille Cedex, France.
Montpellier-Pala
Claire
C
Cocquerel
Laurence
L
Wychowski
Czeslaw
C
Penin
François
F
Dubuisson
Jean
J
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
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D014764:Viral Proteins; C088369:p7 protein, Hepatitis C virus; D000086082:Viroporin Proteins