1887

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 () 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 2A 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 2A and human eIF-4G. Initially, 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 2A in an inducible manner does not affect p150, but led to the efficient cleavage of human eIF-4G in yeast cells. Induction of 2A was detrimental to luciferase synthesis either from luc mRNA or Leader-luc mRNA irrespective of the presence or absence of human eIF-4G. 2A blocked luciferase expression at the transcriptional level. Finally, the effects of 16 point mutations of poliovirus 2A on luciferase expression and human eIF-4G cleavage were analysed. Only those 2A variants that generate viable polioviruses actively cleave eIF-4G in yeast.

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1998-11-01
2021-10-27
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