Alternate Translation Occurs within the Core Coding Region of the Hepatitis C Viral Genome

Abstract

The majority of hepatitis C virus (HCV) isolates contain an open reading frame (ORF) overlapping with the core coding sequences in the ؉1 frame, which was assumed to be untranslated. We present evidence supporting the expression of this ORF (designated core؉1 ORF) via novel translation mechanisms. First, fusion of the luciferase gene with the HCV-1 core؉1 ORF followed by in vitro translation resulted in the synthesis of a chimeric protein (core؉1-luciferase) that exhibited ϳ54% luciferase activity relative to the positive control (coreluciferase). Second, antisera raised against two different synthetic core؉1 peptides recognized the previously identified p16 (but not p21) core protein band expressed from HCV-1, indicating the presence of epitopes from the core؉1 ORF within the p16 protein. Third, HCVpositive sera specifically recognized lysates of Escherichia coli cells expressing recombinant core؉1 protein, suggesting the presence of anti-core؉1 antibodies in HCV-infected patients. Finally, luciferase tagging experiments designed to assess for ؊1 frameshifting combined with site-directed mutagenesis experiments supported the presence of ؉1/؊1 ribosomal frameshift translation mechanisms within the core coding region. In conclusion, our data provide evidence for novel translation mechanisms within the core coding region and demonstrate the expression of the core؉1 ORF, at least for some HCV isolates.

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

“…Moreover, HCV-1 codons 8-11 have been shown to direct both 22/11 and 21/12 programmed ribosomal frameshifting (27,28). However, our expression studies in RRL with the HCV-1a (H) isolate, in which the A-rich region is disrupted by a G and a C at the fourth and tenth positions, respectively (A AAG AAA AAC), showed that HCV-1a (H) did not produce p16 core11/F protein (18). This suggests that the efficient synthesis of core11/F protein is directly dependent on the presence of the 10As.…”

“…Since its discovery, several studies have shown that the core11/ARFP/F protein is specifically recognized by antibodies in serum (17)(18)(19)(33)(34)(35)(36)(37)(38)(39)(40) and by the memory T cells (34,36) of HCV-infected patients, providing evidence that the core11 ORF is produced during natural HCV infection in humans. However, initial studies gave variable frequencies for the detection of core11-humoral immune responses in HCV-infected individuals.…”

“…Extensive site-directed mutagenesis within the core/core11 coding region (18) and radioactive sequencing of the protein synthesized in vitro (19) showed that the production of this novel 16 kDa protein resulted from the initiation of translation at the AUG initiator codon of the polyprotein sequence, followed by a 22/11 ribosomal frameshift in an adenosine (A)-rich region located within core codons 8-11. The p16 protein is known as ARFP (alternative reading frame protein) (17), F (for frameshift) (19) or core11 (to indicate the position of the new open reading frame) protein (18,20).…”

“…This suggests that transcriptional slippage by T7 or NS5B polymerases may account for synthesis of the core11/F protein in T7-based expression systems. However, our initial expression studies in RRL, that provided the first evidence of 22/11 frameshifting within the core coding sequence at the stretch of 10 adenosine residues, were based on SP6-driven transcripts (18).…”

“…The p16 protein is known as ARFP (alternative reading frame protein) (17), F (for frameshift) (19) or core11 (to indicate the position of the new open reading frame) protein (18,20).…”