Characterization of the expression of the hepatitis C virus F protein.
Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is approximately 9.6 kb in length and encodes a polyprotein that is proteolytically cleaved to generate at least 10 mature viral protein products. Recently, a new protein, named F, has been described to be expressed through a ribosomal frameshift within the capsid-encoding sequence, a mechanism unique among members of the family Flavidiridae: Here, expression of the F protein was investigated in an in vitro transcription/translation assay. Its expression in mammalian cells was confirmed using specific recombinant vaccinia viruses; under these conditions, protein expression is dependent on the HCV IRES. The F protein was tagged with firefly luciferase or the Myc epitope to facilitate its identification. Ribosomal frameshifting was dependent on the presence of mutations in the capsid-encoding sequence. No frameshifting was detected in the absence of any mutation. Furthermore, analysis of the F protein in time-course experiments revealed that the protein is very unstable and that its production can be stabilized by the proteasome inhibitor MG132. Finally, indirect immunofluorescence studies have localized the F protein in the cytoplasm, with notable perinuclear detection.
Roussel
Juliette
J
Laboratoire de Virologie, Centre Hospitalier Universitaire-Hôpital Sud, 80054 Amiens Cedex, France.|CNRS-UPR 2511, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France.
Pillez
André
A
CNRS-UPR 2511, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France.
Montpellier
Claire
C
CNRS-UPR 2511, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France.
Duverlie
Gilles
G
Laboratoire de Virologie, Centre Hospitalier Universitaire-Hôpital Sud, 80054 Amiens Cedex, France.
Cahour
Annie
A
CERVI (Virologie), UPRES EA 2387, Hôpital Pitié-Salpêtrière, 75651 Paris Cedex 13, France.
Dubuisson
Jean
J
CNRS-UPR 2511, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France.
Wychowski
Czeslaw
C
CNRS-UPR 2511, IBL/Institut Pasteur de Lille, 59021 Lille Cedex, France.
D001483:Base Sequence; D002460:Cell Line; D018965:Frameshifting, Ribosomal; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011995:Recombination, Genetic; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D007976:Leupeptins; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde