A variety of approaches have been devised to express PV 2A pro in order to cleave eIF4G in culture cells or in cellfree systems. Of these approaches, the most straightforward system has been the addition of the purified PV 2A pro , usually as a hybrid protein such as MBP-2A pro , to cellfree systems such as rabbit reticulocyte lysates (RRLs), HeLa and Krebs-2 extracts [76, 118, [124] [125] [126] [127] . In this sense, the addition of about 1 to 5 μg MBP-2A pro suffices to hydrolyze eIF4G in those cell-free systems [76, [125] [126] [127] . An alternative method to cleave eIF4G in an in vitro system is the translation of an mRNA encoding PV 2A pro in translation competent extracts [50] . This assay has the advantage of providing genuine and freshly made PV 2A pro , leading to total cleavage of eIF4G in the test tube after several minutes of translation. Many different approaches have been explored in culture cells, the most popular being transfection of plasmids encoding PV 2A pro in different eukaryotic cell types [75, 76, [128] [129] [130] [131] [132] [133] [134] . Several plasmids have been utilized in this respect, and perhaps the most successful one is pTM1-2A, which is transfected in mammalian cells that transiently express T7 RNA polymerase by infection with a recombinant vaccinia T7 virus [76, 130, 131, 133, 134] . The amount of protease synthesized in this system is similar to that found in PV-infected cells at late times of infection, but these amounts are reached 1-2 hours after transfection. Similar results have been obtained in cells constitutively expressing T7 polymerase, which comprises a less pleiotropic system, because vaccinia virus proteins are not expressed (unpublished data). Since PV 2A pro targets a number of different cellular proteins, which affect several cellular functions depending on the amount of protease synthesized (see below), in some instances, it is useful to express 2A pro at low levels. We have explored many alternative methods trying to get a system that allows us to control the levels of PV 2A pro into the cells. Novoa and colleagues developed a protocol based on the addition of hybrid proteins bearing PV 2A pro . These recombinant proteins enter into the cytoplasm on cell membrane permeabilization by different methods such as addition of MBP-2A pro mixed with replicationally inactive chicken adenovirus particles [135] . Cleavage of eIF4G following these protocols takes place after incubation for 8-10 hours, suggesting that the amount of protease internalized is probably low but sufficient to hydrolyze eIF4GI in virtually all culture cells [136] . Probably one of the most attractive systems is a stable cell line that inducibly express PV 2A pro , obtained in two different laboratories including ours [137, 138] . In these cell lines, PV 2A pro is synthesized when tretracycline is removed from the culture medium, leading to low expression of PV 2A pro that induces efficient cleavage of eIF4G after 13 h post induction correlating with a potent inhibition of cellular translation. Finally, long term expression of PV 2A pro in Tet off cell lines triggers apoptosis [12, 137, 138] . The main drawback of this cell line is the low PV 2A pro escape under repression conditions that gives rise to a basal cytotoxicity. Probably, the most efficient method is electroporation of an mRNA encoding 2A pro under the control of EMCV leader sequence (IRES-2A) [95] . The biggest advantage of this method is the capacity to regulate levels of 2A pro expression by controlling the amounts of IRES-2A transfected. For example, electroporation of 9 μg of IRES-2A into ∼ 1.5 · 10 6 HeLa cells leads to total cleavage of both eIF4GI and eIF4GII in only 2 h, resulting in an almost complete shutoff of cellular protein synthesis. In contrast, electroporation of low amounts of IRES-2A (1 μg) into HeLa cells induces efficient cleavage of eIF4GI, whereas eIF4GII remains largely intact. Therefore, 9-fold more IRES-2A mRNA is required to cleave eIF4GII compared to eIF4GI [95] . Based on the IRES-2A mRNA electroporation method we were able to induce the differential proteolysis of eIF4GI and eIF4GII in a timeand dose-dependent manner. Kinetics of protein synthesis shutoff and eIF4GII cleavage is closely correlated in HeLa cells, resembling what was found in PV-infected cells [122] .