Figure 4

Sensitivity of human and zebrafish PKR to inhibition by vIF2α K3 and E3. Plasmids expressing VACV K3L (pC140), RCV-Z vIF2α (pC3853), or VACV E3L (p2245) under the control of a yeast GAL-CYC1 hybrid promoter, or the empty vector pEMBLyex4, were introduced into isogenic yeast strains having either an empty vector (A, J673), a GAL-CYC1-human PKR construct (B, J983), or a GAL-CYC1-zebrafish PKR construct (C, J944) integrated at the LEU2 locus. The indicated transformants were streaked on SC-Gal medium where expression of both PKR and the viral proteins was induced, and incubated at 30°C for 4 days. Results shown are representative of 4 independent transformants for each plasmid. (D) Transformants described in panels A-C were grown in liquid SC-Gal medium for 13 hours, then whole cell extracts were obtained from equal numbers of cells and subjected to SDS-PAGE followed by immunoblot analysis. Following transfer to nitrocellulose membranes, the upper halves of the blots were probed with phosphospecific antibodies against Thr446 in human PKR (second panel from top), then stripped and probed with anti-Flag tag antibodies which detect Flag-tagged human and zebrafish PKR (top panel). The lower part of the blot was incubated with phosphospecific antibodies against Ser51 in eIF2α (eIF2α-P; third panel from top), then stripped and probed with polyclonal antiserum against total yeast eIF2α. Lane 9 contains protein extracts from the vector (pEMBLyex4) transformed control strain (J673, panel A). The ratios between phosphorylated eIF2α and total eIF2α converted to percentages are shown below.