Protein kinase R reveals an evolutionary model for defeating viral mimicry

Abstract

Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis and cytoskeletal dynamics. Other mimics interfere with immunity. Poxviruses encode K3L, a mimic of eIF2alpha, which is the substrate of protein kinase R (PKR), an important component of innate immunity in vertebrates. The PKR-K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2alpha) while avoiding rapidly evolving substrate mimics such as K3L. Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under intense episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2alpha recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, although it can seem that pathogens gain insurmountable advantages by mimicking cellular components, host factors such as PKR can compete in molecular 'arms races' with mimics because of evolutionary flexibility at protein interaction interfaces challenged by mimicry.

Figure 1. Widespread positive selection shaped PKR throughout primate evolution

(a) PKR was sequenced from simian primates that together represent more than 30 million years of divergence. dN/dS values along each branch of the phylogeny are listed, and those with dN/dS>1 are highlighted in red. Branches with bold lines, overlapping the set in red, indicate lineages found to be under positive selection by complementary model fitting analysis (also see Table S6). Values in parentheses are shown for branches where no synonymous changes were observed (S=0) and indicate the number of non-synonymous changes (N). (b) Sites under positive selection (red) are mapped onto a ribbons representation of the PKR kinase domain (blue) / eIF2α (green) complex (PDB code: 2A1A). The active site of PKR is shown in orange and a large portion of the β4-β5 loop (dashed blue line) is invisible from the structure deduced from the co-crystal for technical reasons. Residues under positive selection near the interface of PKR with eIF2α and K3L are noted in the β4-β5 loop (Thr336, Asp338, Ser344, Ser351) and the αD (Gln376, Lys380) and αG (Phe489, Ser492, Thr496) helices. (c) Plasmids encoding PKR variants from a panel of primates under pGal were introduced into yeast strains HM3 (eIF2α), HM2 (eIF2α and HA-vaccinia K3L), and J223 (eIF2α-S51A). Ten-fold serial dilutions of transformants were spotted on plates containing either glucose or galactose (see Full Methods). Immunoblot analysis of PKR (top panel) and HA-K3L (bottom panel) is also shown (see Full Methods). For AGM, resistance to K3L might reflect differences in PKR expression in yeast. (d) Primary fibroblasts from the indicated primates were infected with WT or ΔK3L vaccinia virus in triplicate (moi=0.001). Virus production was assessed three days post infection by titering cell lysates. The significance of WT versus ΔK3L is indicated (Student’s t-test; bars show s.d.). Minor variations of this experiment (not shown) revealed that ΔK3L infections typically produced ~5-fold less virus than wildtype virus in gibbon cells.

Figure 2. Distinct surfaces of the PKR kinase domain are critical to K3L resistance

(a) Plasmids encoding gibbon PKR alleles with substitutions in the αG helix were introduced into yeast strains HM3 (eIF2α alone) and HM1 (eIF2α and K3L). Ten-fold serial dilutions of transformants are shown. Corresponding immunoblot analysis is also shown using antibodies against PKR (top panel) and K3L (bottom panel). (b) A ribbon representation of the PKR/eIF2α complex highlighting the association of side chains of residues under positive selection with side chains of eIF2α. Phe489, Ser492, and Thr496 form a face of the αG helix directly interacting with eIF2α. (c) Plasmids encoding gibbon and orangutan PKR alleles with substitutions in the αE helix were introduced into yeast strains HM3 and HM1. Ten-fold serial dilutions of transformants are shown along with corresponding immunoblot analysis. (d) Residues under positive selection (Gln376 and Lys380) and residue Leu394 from a ribbon representation of human PKR and eIF2α are shown. (e) A schematic depicting that single substitutions in either the αE and αG helices can confer resistance against vaccinia K3L to gibbon PKR.

Figure 3. PKR chimeras reveal masking of K3L sensitivity by Leu394

(a) Ten-fold serial dilutions of transformants expressing alleles of human PKR with combinations of substitutions in the αE and αG helices are shown along with corresponding immunoblot analysis. (b) Phenotype ‘cubes’ summarizing the K3L susceptibility of alleles with all combinations of substitutions between human and gibbon PKR at positions 394, 489, and 492 from Figures 2a, 3a and S5. Red and blue dots indicate resistance and sensitivity to K3L respectively. With the exception of F-F-A, which shows some measure of resistance to K3L in the human background (indicated by the red crescent), each set of substitutions have similar phenotypes in the human and gibbon backgrounds. Each single substitution in wildtype human PKR results in a variant still resistant to K3L, while in two of three cases gibbon PKR becomes resistant (indicated by arrows). (c) Sequence alignments of the αG helix for each member of the eIF2α kinase family from several mammals highlights the conservation of this region compared to rapid evolution of PKR (black arrowheads indicate residues of the αG helix under positive selection in PKR). The frequency of substitutions among the panel at each position is indicated by a color code (yellow for a single substitution, orange for a second, red for a third, and blue for a fourth) with human sequence as a reference. Residues making contacts with eIF2α are indicated with lines below the PKR alignment.