Expression studies of the HCV-1a core+1 open reading frame in mammalian cells.
The hepatitis C virus (HCV) genome possesses an open reading frame overlapping the core gene in the +1 frame (core+1 ORF). Initial studies, mainly in rabbit reticulocyte lysates, indicated that the HCV-1 core+1 ORF is expressed by a -2/+1 frameshift at codons 8-11 during translation elongation of the viral polyprotein, resulting in a protein known as alternative reading frame protein (ARFP), frameshift (F), or core+1. However, subsequent investigation, based on reporter constructs carrying portions of the core+1 ORF, suggested the function of alternative mechanisms for core+1 expression in mammalian cells, including translation initiation from internal codons 85/87 or 26. Because results from these studies have been variable, we sought to re-evaluate expression of the core+1 ORF using constructs carrying the complete core+1 coding sequence fused to GFP or LUC. We showed here that codons 85/87 serve as the predominant initiation sites for internal translation initiation of core+1 ORF in Huh-7 and Huh-7/T7 mammalian cells, which support nuclear or cytoplasmic transcription, respectively. We also showed that internal translation initiation can occur concomitantly with the expression of the core+1/F protein that is produced artificially in Huh-7 or naturally in Huh-7/T7 cells. Furthermore, translation of core+1 ORF is not significantly affected by the presence of the HCV IRES element. The core+1/S-GFP protein is cytoplasmic and exhibits an ER distribution similar to that of the core+1/F-GFP protein.
Vassilaki
Niki
N
Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, 11521 Athens, Greece.
Boleti
Haralabia
H
Mavromara
Penelope
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D045744:Cell Line, Tumor; D003062:Codon; D018387:Codon, Initiator; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D018387:Codon, Initiator; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus; D049452:Green Fluorescent Proteins; D008156:Luciferases