The Yeast Saccharomyces cerevisiae as a Genetic System for Obtaining Variants of Poliovirus Protease 2A

Abstract

pro 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 2A pro 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 2A pro mutant sequences, including 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. cerevisiae represents a useful system for obtaining poliovirus 2A pro variants that may provide further insight into the role of this protease during the poliovirus replication cycle.

Mutational Analysis of Poliovirus 2Apro

“…Poliovirus type 1 (Mahoney strain) was obtained and used as described previously (29). Poliovirus mutants bearing substitutions in 2A pro , named as M3 (SS6F), M6 (D135N), and M7 (V119M) were obtained as described previously (40).…”

“…Mutants N18K, A22T, L39F, C55Y, G60R, S66F, G100N, G110S, V119M, G121E, A125V, G126D, S134L, and D135N were obtained by random mutagenesis and genetic selection in yeast as indicated (40).…”

“…S66F (M3), D135N (M6), and V119M (M7), were assayed for their effect on luciferase gene expression. These poliovirus variants, which have been characterized in detail recently (40), are viable but show defects in the transactivation of translation by 2A pro . The three poliovirus mutants tested cleaved eIF-4G and shut down host translation (Fig.…”

“…2A pro mutants with deletions at the amino terminus (⌬1-22), at the carboxyl terminus (⌬101-146; ⌬124 -146), as well as internally (⌬66 -97) were generated. In addition, 17 point mutations in 2A pro were obtained, some of them by site-directed mutagenesis (Y88S, Y88P, and Y88L) and the rest using a recently described genetic system to select cytotoxic-deficient 2A pro mutants in yeast cells (40). Mutant 2A-1 contains a leucine insertion at position 103 as described (44 (Fig.…”

A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2A pro : Effects on Cellular and Viral Gene Expression

“…The potential cellular toxicity of the individual expression of 2A pro has been analyzed in different cellular systems, including bacteria where this protease was nontoxic (4,48), yeast (7,8,31), and mammalian cells. A variety of expression systems have been examined in cultured cells.…”