Differences in the expression of the hepatitis C virus core+1 open reading frame between a nuclear and a cytoplasmic expression system

The hepatitis C virus (HCV) genome possesses an open reading frame (ORF) overlapping the core gene at + 1 nucleotide (core+ 1 ORF). Initial in vitro studies suggested that the core+ 1 ORF is translated by a ribosomal -2/+1 frameshift mechanism during elongation of the viral polyprotein. Recent studies, however, based on transfection of mammalian cells with reporter constructs have shown that translation of the core + 1 ORF is mediated from internal core + 1 codons. To resolve the apparent discrepancies associated with the mechanism of core+ 1 translation, we examined the expression of the HCV-1 and HCV-1 a (H) core+ 1 ORF in a cytoplasmic transcription system based on Huh-7/T7 cells that constitutively synthesize the T7 RNA polymerase in comparison to that in Huh-7 cells. We showed that the efficiency of both the -2/+ 1 and -1/+2 frameshift events operating at the HCV-1 core coclons 8-11 is significantly enhanced in the Huh-7/T7 cytoplasmic transcription system and is dependent on the presence of the consecutive adenine (A) residues within core codons 8-11. In contrast, internal translation initiation at core+ 1 codons 85/87 occurs in both the nuclear and cytoplasmic transcription systems and is not repressed by the ribosomal frameshifting event. Finally, although core + 1 codons 85/87 is the most efficient site for internal initiation of core + 1 translation, it may not be unique, as additional internal core+ 1 codon(s) appear to drive translation at low levels.