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.
Boumlic
Anissa
A
Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas Sophias Ave, 11521 Athens, Greece.
Vassilaki
Niki
N
Dalagiorgou
Georgia
G
Kochlios
Emmanouil
E
Kakkanas
Athanassios
A
Georgopoulou
Urania
U
Markoulatos
Panagiotis
P
Orfanoudakis
Georges
G
Mavromara
Penelope
P
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