Translation of the F protein of hepatitis C virus is initiated at a non-AUG codon in a +1 reading frame relative to the polyprotein

Abstract

The hepatitis C virus (HCV) genome contains an internal ribosome entry site (IRES) followed by a large open reading frame coding for a polyprotein that is cleaved into 10 proteins. An additional HCV protein, the F protein, was recently suggested to result from a +1 frameshift by a minority of ribosomes that initiated translation at the HCV AUG initiator codon of the polyprotein. In the present study, we reassessed the mechanism accounting for the synthesis of the F protein by measuring the expression in cultured cells of a luciferase reporter gene with an insertion encompassing the IRES plus the beginning of the HCV-coding region preceding the luciferase-coding sequence. The insertion was such that luciferase expression was either in the +1 reading frame relative to the HCV AUG initiator codon, mimicking the expression of the F protein, or in-frame with this AUG, mimicking the expression of the polyprotein. Introduction of a stop codon at various positions in-frame with the AUG initiator codon and substitution of this AUG with UAC inhibited luciferase expression in the 0 reading frame but not in the +1 reading frame, ruling out that the synthesis of the F protein results from a +1 frameshift. Introduction of a stop codon at various positions in the +1 reading frame identified the codon overlapping codon 26 of the polyprotein in the +1 reading frame as the translation start site for the F protein. This codon 26(+1) is either GUG or GCG in the viral variants. Expression of the F protein strongly increased when codon 26(+1) was replaced with AUG, or when its context was mutated into an optimal Kozak context, but was severely decreased in the presence of low concentrations of edeine. These observations are consistent with a Met-tRNAi-dependent initiation of translation at a non-AUG codon for the synthesis of the F protein.

The HCV ARFP/F/core+1 protein: Production and functional analysis of an unconventional viral product

“…However, RRL did not support the synthesis of this form of core11 protein (24). Consistent with our data, Baril and Brakier-Gingras also reported that internal translation initiation, rather than frameshift, was the main mechanism of core11 protein synthesis in cultured human cells and showed that the frameshift mechanism required the presence of the 10 consecutive adenosines within codons 8-11 (21). However, codon 26 was mapped as the internal translation initiation site.…”

“…However, there is no consensus on the nature of the core11 protein, as alternative translation initiation sites and different mechanisms for core11 ORF expression have been described (21)(22)(23)(24) (Fig. 1).…”

Transcriptional slippage prompts recoding in alternate reading frames in the hepatitis C virus (HCV) core sequence from strain HCV-1

“…Sequence analysis of the minor quasispecies from infected patients with high levels of antibodies against frameshifted proteins would help to address this question. An alternative explanation for the high prevalence of anti-ARFP antibodies would be expression of the core protein +1 ORF by alternative mechanisms such as internal initiation, as described previously (Vassilaki & Mavromara, 2003;Baril & Brakier-Gingras, 2005).…”

“…Additional studies have revealed that this A-rich sequence is also able to induce -1 frameshifting (Choi et al, 2003). More recently, it has been shown that internal initiation near codons 85/87 (Vassilaki & Mavromara, 2003) or at codon 26 (Baril & Brakier-Gingras, 2005) in the core coding sequence may generate products in the +1 reading frame. Previously, we demonstrated another pathway for expression of alternative reading frame products from a genotype 1b sequence (GenBank accession no.…”

Development of a Recombinant Based ELISA using Specific Antibodies to F Protein in HCV Chronically Infected Patients-A Seroprevalence Study

“…HCV is a major cause of chronic liver disease which chronically infects more than 170 million persons worldwide. The prevalence of HCV infection varies throughout the world affecting an estimated 3% of the world's population, with rates reaching more than 10% in some countries (1)(2)(3). In Iran, the prevalence of anti-HCV was reported to be 0.5% in general population (4).…”