As observed previously PKR autoinhibits its own expression in yeast [34, 40, 45]. Presumably PKR phosphorylation of eIF2α leads to suppression of total protein synthesis including PKR expression. Accordingly, inhibition of PKR by the viral inhibitors restores protein synthesis and leads to higher PKR levels. Taken LXH254 together, the results of the PKR expression and selleck screening library eIF2α phosphorylation studies demonstrate that vIF2α can effectively inhibit eIF2α phosphorylation by human and zebrafish PKR. In the presence of effective eIF2α phosphorylation inhibitors, PKR migrated faster on SDS-PAGE than

in the controls (Figure 4D, top panel, lanes 2-4 versus 1 and lanes 7-8 versus 5). This might have been caused by inhibition of PKR autophosphorylation. To examine PKR autophosphorylation, we probed the Western blots with a phospho-specific antibody that recognizes human PKR phosphorylated on Thr446. High levels of Thr446 phosphorylation were detected in the absence of inhibitors and when either K3 or vIF2α were present. Thr446

phosphorylation was effectively inhibited in the presence of E3 (Figure 4D, second panel, lanes 1-4). These results indicate that K3 and vIF2α are unable to block Thr446 phosphorylation and are consistent with previous findings that K3 binding to PKR is dependent on Thr446 phosphorylation [18]. Presumably vIF2α, like K3, binds to PKR following autophosphorylation on Thr446 and blocks subsequent autophosphorylation events that lead to altered mobility of PKR on SDS-PAGE. Zebrafish PKR was not detected with the antibody directed against SB273005 Thr446-phosphorylated human PKR (Figure 4D, second panel, lanes 5-8). This was expected because

of the strong sequence divergence between human and zebrafish PKR surrounding the phosphorylation site [27]. Finally, using yeast growth rate assays as described above, vIF2α was found to inhibit, at least partially, both Xenopus laevis PKR1 and zebrafish Urease PKZ (data not shown). However, precise determination of PKR1 and PKZ sensitivity to vIF2α inhibition will depend on the ability to obtain yeast strains expressing the appropriate level of each kinase. In order to test which domains of vIF2α are important for PKR inhibition we tested various vIF2α deletion mutants for their ability to inhibit PKR activity. Additionally, the C-terminus of RCV-Z vIF2α was extended to match the length of ATV vIF2α (see Figure 1). For the latter constructs, the 26 C-terminal amino acids found in ATV vIF2α that are not in RCV-Z vIF2α due to an early termination codon were appended to the C-terminus of RCV-Z vIF2α (vIF2α+26C, Figure 5A). None of the vIF2α constructs led to a growth defect in the control strain not expressing PKR (Figure 5B). In a zebrafish PKR-expressing strain, wild-type vIF2α, vIF2α+26C, and vIF2αΔ59C (lacking the C-terminal 59 amino acids) led to comparable inhibition of PKR toxicity (Figure 5C, sectors 2-4 versus 1).