Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A(pro) modulating the alternative splicing of pre-mRNAs. Expression of 2A(pro) potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A(pro) abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A(pro), leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A(pro) on splicing is to selectively block the second catalytic step. (C) 2011 Elsevier Inc. All rights reserved.