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A structural and functional analysis of opal stop codon translational readthrough during Chikungunya virus
Raymond Li1, Kaiwen Sun1,2, Andrew Tuplin1
1School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, UK.
The Journal of General Virology
|October 20, 2023
Summary
Chikungunya virus (CHIKV) replication requires translational readthrough of a stop codon in nsP3. This study found that while opal and amber codons are tolerated in mammalian cells, ochre is not, with mosquito cells tolerating all three.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes mosquitoes.
- CHIKV's RNA genome encodes non-structural (nsP) and structural proteins.
- Translational readthrough of a stop codon is essential for producing full-length nsP3 and nsP4.
Purpose of the Study:
- To investigate the role of the nsP3 stop codon in CHIKV replication.
- To determine the impact of different stop codons on CHIKV replication in mammalian and mosquito cells.
- To explore the structural requirements for translational readthrough.
Main Methods:
- Site-directed mutagenesis to replace the arginine codon with opal, amber, and ochre stop codons.
- Replication assays using subgenomic replicons and infectious CHIKV.
- SHAPE analysis to interrogate RNA structure.
- Mutagenesis to probe stem-loop structures.
Main Results:
- Opal and amber stop codons were tolerated in mammalian cells, but ochre was not.
- All three stop codons were tolerated in mosquito cells.
- A putative stem-loop structure 3' of the stop codon was not required for translational readthrough.
Conclusions:
- The nsP3 stop codon plays a critical role in CHIKV replication.
- Cell-type specific differences exist in the tolerance of stop codons.
- Factors other than the identified stem-loop structure facilitate CHIKV translational readthrough and replication.
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