A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting capdependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.Infection with poliovirus results in a dramatic shutoff of host protein synthesis that is followed by a selective and efficient translation of the viral mRNA (9). Cellular mRNAs contain a 5Ј-terminal cap structure which plays a pivotal role in the process of initiation of their translation (40). In contrast, poliovirus mRNA is uncapped (15,32), and its translation is initiated by an alternative mechanism that involves direct landing of the ribosomes at an internal site termed internal ribosome entry site (IRES) (19,36). An early event occurring during poliovirus infection is cleavage of the eukaryotic translation initiation factor 4G (eIF4G) by the viral 2A protease (10). Since eIF4G acts as an important mediator that bridges eIF3 (which is complexed to the 40S ribosomal subunit) and the cap binding protein eIF4E (18,23), the translation of cap-dependent mRNAs is selectively inhibited (10, 34). Picornavirus RNAs utilize the C-terminal fragment of eIF4G for translation (35,38). A recent study suggested that the translational inhibitory effect of poliovirus infection may under certain conditions trigger apoptotic cell death (44). Since infection with the entire virus is likely to complicate any interpretation concerning the contribution of individual viral proteins to cellular effects, it was important to express individual viral genes in cells. In this respect, the viral 2A protease is of particular interest. Since expression of the 2A protease is likely to be toxic to cells, all previous attempts to express it were by means of transient transfection (2, 33, 39). However, transient transfections are subjected to variations in the proportion of successfully transfected cells, and therefore the effects are measured in a mixed cell population. We have therefore efficiently expressed the poliovirus 2A protease in an inducible ...