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Unusual multiple recoding events leading to alternative forms of hepatitis C virus core protein from genotype 1b
Steeve Boulant1, Michel Becchi, François Penin
1Laboratoire 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.
Insights
Hepatitis C virus (HCV) core protein exhibits alternative forms due to frameshift mutations in genotype 1b. These genotype-specific recoding events may influence HCV pathogenesis and persistent infection.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Hepatitis C virus (HCV) core protein is crucial for viral particle formation and pathogenesis.
- Understanding HCV core protein variations is key to deciphering persistent infection mechanisms.
Purpose of the Study:
- To identify and characterize alternative forms of genotype 1b HCV core protein.
- To investigate the occurrence and relevance of RNA recoding events in HCV genotypes.
Main Methods:
- Expression of HCV core protein segments in Escherichia coli.
- Analysis using proteolysis, mass spectrometry, and amino acid sequencing.
- In vitro and cell-based assays to confirm recoding events in eukaryotic systems.
Main Results:
- Identified alternative HCV core protein forms resulting from +1 frameshifts at codon 42 and rephasing events.
- Confirmed recoding events in eukaryotic translation systems (reticulocyte lysate and cultured cells).
- Demonstrated differential reactivity of patient sera against +1 open reading frame peptides, highlighting genotype-specific features.
Conclusions:
- Distinct RNA recoding events occur in genotype 1a and 1b HCV.
- These genotype-dependent recoding events likely contribute to HCV pathogenesis and infection persistence.
Abstract:
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.