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Co-expression of human eIF-4G and poliovirus 2Apro in Saccharomyces cerevisiae: effects on gene expression

A Barco1, L Carrasco

  • 1Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, Spain.

The Journal of General Virology
|November 20, 1998
PubMed

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.

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