Two Alternative Translation Mechanisms Are Responsible for the Expression of the HCV ARFP/F/Core+1 Coding Open Reading Frame

Abstract

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.

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

“…Thus, the initiation of translation of the core11 ORF at internal codons 85/87 resulted in the synthesis of another form of core11 protein shorter than that produced by the 11 frameshift mechanism (F) and known as core11/S (short). 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, 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).…”

“…These discrepancies were resolved by expressing a luciferasetagged core11 ORF (carrying the first 95 core11 codons) from HCV-1 and HCV-1a (H) in transfected mammalian cells in culture (24). These studies revealed that both HCV-1 and HCV-1a isolates support expression of the core11 ORF.…”

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.…”