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Co-expression of human eIF-4G and poliovirus 2Apro in Saccharomyces cerevisiae: effects on gene expression
1Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, Spain.
Abstract:
The poliovirus 5' untranslated region (5' UTR) confers on mRNAs the capacity to be translated by internal initiation. The functionality of this RNA motif has been tested in yeast cells (Saccharomyces cerevisiae) using luciferase (luc) as a reporter gene. Although some luciferase is synthesized from luc mRNA containing the poliovirus 5' UTR (Leader-luc mRNA), much more luciferase is synthesized in cells that express luc mRNA devoid of the poliovirus 5' UTR. Since poliovirus 2Apro enhances the translation of Leader-luc mRNAs after eIF-4G cleavage in mammalian cells, yeast cells were produced that synthesize three heterologous proteins, luciferase, poliovirus 2Apro and human eIF-4G. Initially, S. cerevisiae cells constitutively expressing human eIF-4G were isolated. The human eIF-4G gene does not complement yeast cells defective in the initiation factor counterpart, p150, indicating that the human and yeast eIF-4G are not interchangeable. Expression of poliovirus 2Apro in an inducible manner does not affect p150, but led to the efficient cleavage of human eIF-4G in yeast cells. Induction of 2Apro was detrimental to luciferase synthesis either from luc mRNA or Leader-luc mRNA irrespective of the presence or absence of human eIF-4G. 2Apro blocked luciferase expression at the transcriptional level. Finally, the effects of 16 point mutations of poliovirus 2Apro on luciferase expression and human eIF-4G cleavage were analysed. Only those 2Apro variants that generate viable polioviruses actively cleave eIF-4G in yeast.
Insights
The poliovirus 5' untranslated region (UTR) enables internal translation initiation in yeast. However, poliovirus 2Apro protease inhibits luciferase expression transcriptionally, regardless of eIF-4G cleavage.
Area of Science:
- Molecular Biology
- Virology
- Yeast Genetics
Background:
- The poliovirus 5' untranslated region (UTR) is known to mediate internal translation initiation in eukaryotic mRNAs.
- Investigating the functionality of this RNA motif and its interaction with viral proteins in a heterologous system like yeast can reveal conserved mechanisms.
- Poliovirus 2Apro protease is known to cleave eukaryotic translation initiation factor eIF-4G in mammalian cells, enhancing viral translation.
Purpose of the Study:
- To assess the functionality of the poliovirus 5' UTR in mediating internal translation initiation in yeast (Saccharomyces cerevisiae).
- To investigate the effect of poliovirus 2Apro protease on translation and eIF-4G cleavage in yeast.
- To analyze the impact of human eIF-4G expression and poliovirus 2Apro activity on reporter gene expression in yeast.
Main Methods:
- Luciferase reporter assays were performed in yeast expressing luciferase mRNA with or without the poliovirus 5' UTR.
- Yeast strains were engineered to express heterologous human eIF-4G and poliovirus 2Apro protease under inducible or constitutive promoters.
- Western blotting was used to detect eIF-4G cleavage, and luciferase activity assays quantified reporter gene expression.
Main Results:
- The poliovirus 5' UTR conferred limited internal translation initiation capability in yeast, with higher luciferase expression observed in its absence.
- Expression of poliovirus 2Apro in yeast led to the cleavage of human eIF-4G but inhibited luciferase synthesis at the transcriptional level.
- Mutational analysis revealed that only viable poliovirus 2Apro variants effectively cleaved eIF-4G in yeast, correlating with protease activity.
Conclusions:
- The poliovirus 5' UTR's internal translation initiation function is less efficient in yeast compared to mammalian cells.
- Poliovirus 2Apro protease inhibits gene expression in yeast primarily at the transcriptional level, independent of eIF-4G cleavage.
- The study highlights differences in viral protein function and host-pathogen interactions between yeast and mammalian systems.