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Translation of potato virus S RNA in vitro: evidence of protein processing
1University of Leicester, Department of Botany, UK.
Abstract:
RNA from potato virus S (PVS), a member of the carlavirus group, was translated in vitro in rabbit reticulocyte lysate. Time-course experiments revealed the largest product of Mr 190 kD, decreasing in intensity after 60-min incubations, correlating with the accumulation of a 150-kD peptide. This apparent processing could be blocked by the addition of the amino-acid analogues p-fluorophenylalanine and L-canavanine for phenylalanine and arginine, respectively. L-canavanine also appeared to specifically reduce the quantity of PVS (34 kD) coat protein, concomitant with the synthesis of a 36-kD peptide. Sucrose gradient-fractionated genomic RNA directed the synthesis of predominantly 190-kD peptides that appeared not to be processed in the absence of small molecular weight (subgenomic) RNA products.
Insights
Potato virus S (PVS) RNA translation in vitro produces a large 190 kD protein that is processed into a 150 kD peptide. Amino acid analogues block this processing and affect coat protein synthesis.
Area of Science:
- Molecular biology
- Plant virology
- Protein biochemistry
Background:
- Potato virus S (PVS) is a significant pathogen affecting potato crops worldwide.
- Understanding PVS replication mechanisms is crucial for developing effective disease control strategies.
- Carlavirus gene expression often involves complex post-translational modifications.
Purpose of the Study:
- To investigate the in vitro translation products of Potato Virus S (PVS) RNA.
- To elucidate the processing of PVS-encoded proteins during translation.
- To identify the role of specific amino acids and RNA species in PVS protein synthesis.
Main Methods:
- In vitro translation of PVS RNA using rabbit reticulocyte lysate.
- Time-course experiments to analyze protein product accumulation.
- Use of amino acid analogues (p-fluorophenylalanine, L-canavanine) to inhibit protein processing.
- Sucrose gradient fractionation to separate genomic and subgenomic RNAs.
Main Results:
- The largest in vitro translation product from PVS RNA was a 190 kD peptide, which decreased over time.
- A 150 kD peptide accumulated as the 190 kD product diminished, suggesting proteolytic processing.
- Amino acid analogues blocked the processing of the 190 kD peptide.
- L-canavanine specifically reduced the 34 kD coat protein synthesis and increased a 36 kD peptide.
- Translation of genomic RNA alone produced predominantly unprocessed 190 kD peptides.
Conclusions:
- PVS RNA encodes a large precursor protein (190 kD) that undergoes processing.
- Specific amino acid incorporation and potentially subgenomic RNAs are involved in PVS protein maturation.
- The findings provide insights into the post-translational regulation of carlavirus gene expression.