Two alternative translation mechanisms are responsible for the expression of the HCV ARFP/F/core+1 coding open reading frame.
HCV-1 produces a novel protein, known as ARFP, F, or core+1. This protein is encoded by an open reading frame (ORF) that overlaps the core gene in the +1 frame (core+1 ORF). In vitro this protein is produced by a ribosomal frameshift mechanism. However, similar studies failed to detect the ARFP/F/core+1 protein in the HCV-1a (H) isolate. To clarify this issue and to elucidate the functions of this protein, we examined the expression of the core+1 ORF by the HCV-1 and HCV-1a (H) isolates in vivo, in transfected cells. For this purpose, we carried out luciferase (LUC) tagging experiments combined with site-directed mutagenesis studies. Our results showed that the core+1-LUC chimeric protein was efficiently produced in vivo by both isolates. More importantly, neither changes in the specific 10-A residue region of HCV-1 (codons 8-11), the proposed frameshift site for the production of the ARFP/F/core+1 protein in vitro, nor the alteration of the ATG start site of the HCV polyprotein to a stop codon significantly affected the in vivo expression of the core+1 ORF. Furthermore, we showed that efficient translation initiation of the core+1 ORF is mediated by internal initiation codon(s) within the core/core+1-coding sequence, located between nucleotides 583 and 606. Collectively, our data suggest the existence of an alternative translation initiation mechanism that may result in the synthesis of a shorter form of the core+1 protein in transfected cells.
Vassilaki
Niki
N
Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas Sofias Avenue, Athens, Greece 11521.
Mavromara
Penelope
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D003062:Codon; D018388:Codon, Terminator; D006224:Cricetinae; D004252:DNA Mutational Analysis; D016174:Hepacivirus / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014162:Transfection; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics*
D003062:Codon; D018388:Codon, Terminator; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus; D008156:Luciferases