Co-expression of human eIF-4G and poliovirus 2Apro in Saccharomyces cerevisiae: effects on gene expression.
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.
Barco
A
A
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, Spain.
Carrasco
L
L
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics*; D039603:Eukaryotic Initiation Factor-4G; D015966:Gene Expression Regulation, Fungal; D015967:Gene Expression Regulation, Viral; D005800:Genes, Fungal; D017930:Genes, Reporter; D005814:Genes, Viral; D006801:Humans; D008156:Luciferases; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000235:genetics*; D017955:Poliovirus / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014764:Viral Proteins
D039603:Eukaryotic Initiation Factor-4G; D010448:Peptide Initiation Factors; D014764:Viral Proteins; D008156:Luciferases; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus