Fig 8. Distinct amino acids combinations are responsible for the higher inhibition of CMLV 032 for PKRs from different species.
(A) Seven different mutants of VARV C3L were generated that contain successive combinations of amino acid substitutions that are found in CMLV 032. (B) Crystal structure of VACV K3 [14]. Residues that correspond to amino acids of eIF2α that contact human PKR in a co-crystal structure [52] are shown in purple (residue A41) or red (other residues). Residues that differ between VARV C3 and CMLV 032 are shown in purple (residue A41) or blue (other residues). Residue H47, which has been previously implicated in PKR binding [53], is shown in orange. Residues in eIF2α corresponding to residues 1–3 (asterisk) and 44–52 in K3 were not resolved in the co-crystal structure. (C) Expression of VARV C3 mutants, CMLV 032 and VACV K3 in transfected HeLa-PKRkd cells. Cell lysates from transfected cells were separated on 12% SDS-PAGE gels and analyzed by immunoblot analysis with anti-K3 and anti-β-actin antibodies. (D) HeLa-PKRkd cells were transfected with expression vectors encoding firefly luciferase (0.05 μg), with VARV C3L, the indicated mutants of VARV C3L, or CMLV 032 (0.4 μg), and PKR (0.2 μg) from the indicated species. Luciferase activities were measured 48 hours after transfection and normalized to PKR-only transfected cells to obtain relative luciferase activities. Error bars represent the standard deviations from three independent transfections. Results shown are representative of two independent experiments.