Unusual multiple recoding events leading to alternative forms of hepatitis C virus core protein from genotype 1b.
In addition to its involvement in the formation of the capsid shell of the virus particles, the core protein of hepatitis C virus (HCV) is believed to play an important role in the pathogenesis and/or establishment of persistent infection. We describe here alternative forms of genotype 1b HCV core protein identified after purification of various products of core protein segment 1-169 expressed in Escherichia coli and their analysis by proteolysis, mass spectrometry, and amino acid sequencing. These proteins all result from a +1 frameshift at codon 42 (a different position than that previously reported in genotype 1a) and, for some of them, from a rephasing in the normal open reading frame at the termination codon 144 in the +1 open reading frame. To test the relevance of these recoding events in a eukaryotic translational context, the nucleotide sequences surrounding the two shift sites were cloned in the three reading frames into expression vectors, allowing the production of a C-terminally fused green fluorescent protein, and expressed both in a reticulocyte lysate transcription/translation assay and in culture cells. Both recoding events were confirmed in these expression systems, strengthening the hypothesis that they might occur in HCV-infected cells. Moreover, sera from HCV-positive patients of genotype 1a or 1b were shown to react differently against synthetic peptides encoded in the +1 open reading frame. Together, these results indicate the occurrence of distinct recoding events in genotypes 1a and 1b, pointing out genotype-dependent specific features for F protein.
Boulant
Steeve
S
Laboratoire de Bioinformatique et RMN Structurales, aInstitut de Biologie et Chimie des Proteines, UMR5086 CNRS, Université Claude Bernard Lyon I, 7, Passage du Vercors, 69367 Lyon cedex 07, France.
Becchi
Michel
M
Penin
François
F
Lavergne
Jean-Pierre
JP
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D004926:Escherichia coli / Q000378:metabolism; D016368:Frameshift Mutation; D005838:Genotype; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics*
D003062:Codon; D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D049452:Green Fluorescent Proteins