THE YEAST SACCHAROMYCES-CEREVISIAE AS A GENETIC SYSTEM FOR OBTAINING VARIANTS OF POLIOVIRUS PROTEASE 2A

The inducible expression of poliovirus protease 2A (2A(pro)) blocks th e growth of Saccharomyces cerevisiae. A number of yeast colonies that grow after 2A(pro) induction have been isolated. The majority of these clones express 2A(pro) to control levels, suggesting that their abili ty to divide is not due to the loss of 2A(pro) gene inducibility. The sequences of the 2A(pro) genes isolated from 22 clones were determined . Most of the 2A(pro) sequences from these colonies contain point muta tions in the poliovirus protease. The different variant protease seque nces were transferred to an infectious poliovirus cDNA clone. Translat ion of genomic RNA obtained from these poliovirus mutants in cell-free systems revealed that some of them had defects in their ability to cl eave P1-2A in cis. In addition, several of these variants cleaved the translation initiation factor eIF-4G inefficiently. Transfection of th e RNA generated from the full-length poliovirus genomes mutated in 2A( pro) yielded five viable polioviruses with a small plaque phenotype. T hese 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 2A(pro) mutant sequences, includi ng those that did not yield viable viruses, were cloned in pTM1 vector under a T7 promoter. Only the 2A(pro) variants that have activity to cleave 3CD produced viable poliovirus. Our findings indicate that S. c erevisiae represents a useful system for obtaining poliovirus 2A(pro) variants that may provide further insight into the role of this protea se during the poliovirus replication cycle.