case, addition of IPTG triggered a significant increase on the caspase activation levels with respect to those observed in its corresponding IPTGuntreated control. To analyze in more detail the effect of VP2 expression on cell fate, two sets of HeLa cell cultures were infected with this virus and maintained in medium supplemented with Construction of pcDNA-VP3 A DNA fragment corresponding to the VP3 coding region was generated by PCR from pVOTE.1/VP3 using the primers 59-CGCGAAGCTTATGGGTTTCCCTCACAATCCACGC and 59-GCGCGGATCCTCACTCAAGGTCCTCATCAGAGAC. The resulting fragment contains an artificial ATG codon to allow for initiation of translation. The DNA fragment was purified, restricted with 11904527 HindIII and BamHI and cloned into pcDNA3 previously digested with the same enzymes. The resulting plasmid, pcDNA-VP3, was subjected to nucleotide sequence analysis to assess the correctness of the cloned sequence. Determination of caspase 3/7 activation Determinations were carried out using the Caspase-Glo 3/7 assay kit following 19380825 the protocol recommended by the supplier. Briefly, HeLa cell monolayers grown in 96 well plates were infected at the indicated MOI. At the specified times p.i., 100 ml of Caspase-Glo 3/7 reagent was added to the wells under study. Plates were gently shaken and then incubated in the dark at 20uC for 60 min before recording the luciferase activity using an Orion microplate luminometer. Autoradiography and Western blot analysis For metabolic labeling cell monolayers were washed twice with methionine-free DMEM. Thereafter, cultures were incubated for 30 min with 100 mCi/ml of methionine, washed twice with IBDV VP3 Inhibits PKR-Mediated Apoptosis IPTG. IPTG-treated uninfected cells were used as a control for this experiment. The first 871700-17-3 culture set was used to assess the kinetics of protein synthesis. For this, at different times p.i., ranging from 4 to 32 h, cells were metabolically labeled with methionine for 30 min, and the corresponding samples subjected to SDS-PAGE followed by autoradiography. The second culture set was used to assess the status of selected polypeptides by WB analysis. In agreement to previously reported data, we observed that cells expressing VP2 undergo a potent shut off of protein synthesis evidenced by the steady reduction of methionine incorporation detected in samples collected from 16 h.p.i. onwards. The WB analysis performed with the VP2-specific serum shows that VP2 accumulation is already detectable at 8 h.p.i., and reaches its maximum level at 16 h.p.i.. In addition to the described biochemical changes, cells expressing VP2 
exhibited noticeable morphological alterations, e.g. cell shrinkage and membrane blebbing, typically found in apoptotic cells. One of the most common causes for the inhibition of protein synthesis in virus-infected cells is the phosphorylation of eIF2a. Hence, we analyzed the extent of eIF2a phosphorylation in VP2-expressing cells. As shown in Fig. 1C, whilst the level of total eIF2a remains roughly constant throughout the duration of the experiment, the presence of phosphorylated eIF2a, first noticeable as a very faint band in samples collected at 8 h.p.i., increases with time, thus somehow matching the VP2 expression profile. It has been shown that eIF2a can be phosphorylated by four mammalian serine-threonine protein kinases, namely PKR, general non-derepressible 2 kinase, PKR-like endoplasmic reticulum kinase, and hemin-regulated inhibitor of translation, following diverse stre
