The yeast Saccharomyces cerevisiae as a genetic system for obtaining variants of poliovirus protease 2A.
The inducible expression of poliovirus protease 2A (2Apro) blocks the growth of Saccharomyces cerevisiae. A number of yeast colonies that grow after 2Apro induction have been isolated. The majority of these clones express 2Apro to control levels, suggesting that their ability to divide is not due to the loss of 2Apro gene inducibility. The sequences of the 2Apro genes isolated from 22 clones were determined. Most of the 2Apro sequences from these colonies contain point mutations in the poliovirus protease. The different variant protease sequences were transferred to an infectious poliovirus cDNA clone. Translation of genomic RNA obtained from these poliovirus mutants in cell-free systems revealed that some of them had defects in their ability to cleave P1-2A in cis. In addition, several of these variants cleaved the translation initiation factor eIF-4G inefficiently. Transfection of the RNA generated from the full-length poliovirus genomes mutated in 2Apro yielded five viable polioviruses with a small plaque phenotype. These five polioviruses efficiently cleaved p220 but showed defects in viral protein synthesis, transactivation of a leader-luciferase mRNA, and 3CD cleavage to 3C' and 3D'. All 2Apro mutant sequences, including those that did not yield viable viruses, were cloned in pTM1 vector under a T7 promoter. Only the 2Apro variants that have activity to cleave 3CD produced viable poliovirus. Our findings indicate that S. cerevisiae represents a useful system for obtaining poliovirus 2Apro variants that may provide further insight into the role of this protease during the poliovirus replication cycle.
Barco
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A
Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas-UAM, Universidad Autónoma de Madrid, Canto Blanco, 28049 Madrid, Spain.
Ventoso
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I
Carrasco
L
L
D000595:Amino Acid Sequence; D002455:Cell Division / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017955:Poliovirus / Q000201:enzymology*; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae; D013329:Structure-Activity Relationship; D014764:Viral Proteins
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus