Proteolytically active 2A proteinase of human rhinovirus 2 is toxic for Saccharomyces cerevisiae but does not cleave the homologues of eIF-4 gamma in vivo or in vitro.
During the replication of rhino- and enteroviruses, the translation initiation factor elF-4 gamma is specifically cleaved by the virally encoded 2 A proteinase. This cleavage has been proposed to lead to the inability of the host cell to translate its own capped mRNA and to stimulate internal initiation of protein synthesis from the viral mRNA. However, a direct causal relationship between these effects and 2A proteinase-mediated cleavage of elF-4 gamma has remained difficult to prove, mainly because of the toxicity of the 2A proteinase in mammalian expression systems. As an alternative approach, we placed the cDNA sequences for the human rhinovirus 2 2A proteinase and two mutants defective in proteolytic activity under the control of an inducible yeast Gal1-10 promoter and stably integrated them into the yeast genome. Induction of the wildtype enzyme led to changes in cellular morphology, an inhibition of cell division activity, and finally to cell death. As the yeast homologues of mammalian elF-4 gamma, p150 and p130, were shown to be refractory to cleavage by human rhinovirus 2A proteinase both in vivo and in vitro and the rate of protein synthesis was unaffected, the toxicity of the 2A proteinase toward budding yeast must be due to its interaction with at least one other cellular protein essential for viability.
Klump
H
H
Department of Biochemistry, Medical Faculty, University of Vienna, Austria.
Auer
H
H
Liebig
H D
HD
Kuechler
E
E
Skern
T
T
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism*; D012229:Rhinovirus / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects*; D014764:Viral Proteins
D005656:Fungal Proteins; D010448:Peptide Initiation Factors; D014764:Viral Proteins; C075833:eIF-4; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus