COEXPRESSION OF HUMAN EIF-4G AND POLIOVIRUS 2A(PRO) IN SACCHAROMYCES-CEREVISIAE - EFFECTS ON GENE-EXPRESSION

The poliovirus 5' untranslated region (5' UTR) confers on mRNAs the ca pacity to be translated by internal initiation. The functionality of t his 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 l uc mRNA devoid of the poliovirus 5' UTR. Since poliovirus 2A(pro) enha nces the translation of Leader-luc mRNAs after eIF-4G cleavage in mamm alian cells, yeast cells were produced that synthesize three heterolog ous proteins, luciferase, poliovirus 2A(pro) and human eIF-4G. Initial ly, S. cerevisiae cells constitutively expressing human eIF-4G were is olated. The human eIF-4G gene does not complement yeast cells defectiv e in the initiation factor counterpart, p150, indicating that the huma n and yeast eIF-4G are not interchangeable. Expression of poliovirus 2 A(pro) in an inducible manner does not affect p150, but led to the eff icient cleavage of human eIF-4G in yeast cells. Induction of 2A(pro) w as detrimental to luciferase synthesis either from luc mRNA or Leader- luc mRNA irrespective of the presence or absence of human eIF-4G. 2A(p ro) blocked luciferase expression at the transcriptional level. Finall y, the effects of 16 point mutations of poliovirus 2A(pro) on lucifera se expression and human eIF-4G cleavage were analysed. Only those 2A(p ro) variants that generate viable polioviruses actively cleave eIF-4G in yeast.