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.

Both S1 and helical domains in vIF2± are required for PKR inhibition. (A) Schematic representation of RCV-Z vIF2± constructs tested in yeast growth assays and Western blots analyses. S1 domain (red), helical domain (HD; blue) and C-terminal domain (CTD, yellow) are represented by boxes. Numbers that follow deltas (″) indicate the(number of residues that were deleted from the C- or N-terminus, respectively. The extended C-terminus (26 amino acids) from ATV vIF2α was added to the C-terminus of RCV-Z vIF2α in the constructs with the +26C label. The indicated constructs were introduced into isogenic yeast strains having either an empty vector (B, J673) 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 B-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 half of the blot was probed with anti-Flag tag antibodies, which detect Flag-tagged zebrafish PKR (top panel). The lower part of the blot was incubated with anti-Myc tag antibodies to detect Myc-tagged vIF2α (second panel from top), then stripped and probed with phosphospecific antibodies against Ser51 in eIF2α (eIF2α-P; third panel from top), and finally stripped again and probed with polyclonal antiserum against total yeast eIF2α (bottom panel). The ratios between phosphorylated eIF2α and total eIF2α converted to percentages are shown below.