Internal translation initiation stimulates expression of the ARF/core+1 open reading frame of HCV genotype 1b.
The hepatitis C virus possesses an alternative open reading frame overlapping the Core gene, whose products are referred to as Core+1 or alternative reading frame (ARF) or F protein(s). Extensive studies on genotype HCV-1a demonstrated that ribosomal frameshifting supports the synthesis of core+1 protein, when ten consecutive As are present within core codons 9-11 whereas, in the absence of this motif, expression of the core+1 ORF is mediated mainly by internal translation initiation. However, in HCV-1b, no Core+1 isoforms produced by internal translation initiation have been described. Using constructs which contain the Core/Core+1(342-770) region from previously described HCV-1b clinical isolates from liver biopsies, we provide evidence for the synthesis of Core+1 proteins by internal translation initiation in transiently transfected mammalian cells using nuclear or cytoplasmic expression systems. Site directed mutagenesis analyses revealed that (a) the synthesis of Core+1 proteins is independent from the polyprotein expression, as we observed an increase of Core+1 protein expression from constructs lacking the polyprotein translation initiator, (b) the main Core+1 product is expressed from AUG(85), similarly to the Core+1/S protein of HCV-1a, (c) synthesis of Core+1 isoforms is also mediated from GUG(58) or under certain conditions GUG(26) internal codons, albeit at lower efficiency. Finally, comparable to HCV-1a Core+1 proteins, the HCV-1b Core+1 products are negatively regulated by core expression and the proteaosomal pathway. The expression of Core+1 ORF from HCV-1b clinical isolates and the preservation of translation initiation mechanism that stimulates its expression encourage investigating the role of these proteins in HCV pathogenesis.
Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas Sophias Ave, 11521 Athens, Greece.
Anissa
A
Boumlic
Niki
N
Vassilaki
Georgia
G
Dalagiorgou
Emmanouil
E
Kochlios
Athanassios
A
Kakkanas
Urania
U
Georgopoulou
Panagiotis
P
Markoulatos
Georges
G
Orfanoudakis
Penelope
P
Mavromara
D002460:Cell Line; D018387:Codon, Initiator; D018965:Frameshifting, Ribosomal; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D014176:Protein Biosynthesis; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics
D018387:Codon, Initiator; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus