Rapid evolution of protein kinase PKR alters sensitivity to viral inhibitors

Abstract

Protein kinase PKR (also known as EIF2AK2) is activated during viral infection and phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), leading to inhibition of translation and viral replication. We report fast evolution of the PKR kinase domain in vertebrates, coupled with positive selection of specific sites. Substitution of positively selected residues in human PKR with residues found in related species altered sensitivity to PKR inhibitors from different poxviruses. Species-specific differences in sensitivity to poxviral pseudosubstrate inhibitors were identified between human and mouse PKR, and these differences were traced to positively selected residues near the eIF2alpha binding site. Our findings indicate how an antiviral protein evolved to evade viral inhibition while maintaining its primary function. Moreover, the identified species-specific differences in the susceptibility to viral inhibitors have important implications for studying human infections in nonhuman model systems.

Figure 1. Accelerated evolution of PKR

(a) Maximum likelihood phylogram of the kinase domains (KDs) of PKR, PERK, HRI, GCN2, WEE1, PBK, NEK2 and TTK from human (Homo sapiens, Hs), cattle (Bos taurus, Bt), mouse (Mus musculus, Mm), rat (Rattus norvegicus, Rn), chicken (Gallus gallus, Gg), frog (Xenopus tropicalis, Xt), stickleback fish (Gasterosteus aculeatus, Ga) and zebrafish (Danio rerio, Dr) [-lnL=12690.22265]. The topology of the Bayesian analysis showed comparable results. Posterior probabilities from the Bayesian analysis and bootstrap values from the maximum likelihood analysis are shown below and above major branches, respectively. (b) Relative branch lengths (from (a)) of KDs in the indicated species. Stars denote significant difference from PKR (P<0.001).

Figure 2. Positively selected sites in the PKR kinase domain

(a) Positively selected residues (PSRs) projected on the sequence of the human PKR (hPKR) kinase domain (KD). PSRs in different lineages are color-coded: vertebrates: red; tetrapods: orange; mammals & birds: blue; primates: azure; rodents: magenta; chicken: purple; xenopus: brown; PKZ: green. Sequences of the highly divergent kinase insert between β4 and β5 have been excluded from the analyses and are not shown. (b) PSRs are depicted as sticks and color-coded as above on a cartoon representation of the hPKR-eIF2α (gray) complex (PDB code 2A19). Position of helices αD and αG, which are enriched in PSRs, as well as residues E379 and A488 are indicated. (c) PSRs (blue) are highlighted in a surface representation of the PKR KD. The eIF2α contact site (front, left image) and the dimerization interface (back, right image) are shown. eIF2α contact residues are depicted in yellow; positively selected residues that contact eIF2α are shown in orange. Residues involved in dimerization are colored green; a positively selected residue in the dimer contact region is shown in magenta. Positions of PSRs are color-coded as described in (a).

Figure 3. Altered sensitivity of PKR mutants to poxvirus proteins K3L and E3L

(a) Yeast growth assays. Plasmids expressing WT or the indicated PKR mutants under the control of a galactose-inducible promoter were introduced into a WT strain or strains expressing vacK3L-H47R, varE3L or non-phosphorylatable eIF2α−S51A. Transformants were grown to saturation and 4-μl serial dilutions (of OD₆₀₀ = 1, 0.1, 0.01 and 0.001) were spotted on galactose medium and incubated for 3 days at 30°C. (b) Western blot analyses of whole cell extracts from transformants described in (a) of the vacK3L-H47R strain expressing the indicated PKR mutants. Upper panels were probed with anti-Flag antibody to detect human PKR; middle panels were probed with phosphospecific antibodies against Ser51 in eIF2α; bottom panels were probed with polyclonal antiserum against total yeast eIF2α.

Figure 4. Positively selected residues (PSRs) in PKR that altered sensitivity to poxvirus inhibitors

(a–d) eIF2α contact residues are depicted in yellow; PSRs that when mutated altered sensitivity to poxvirus inhibitors vacK3L-H47R (a), varC3L (b) and varE3L (c) are shown in red (increased resistance), blue (increased sensitivity) and magenta (distinct sets of mutants showed increased resistance or sensitivity). Mutations that had the strongest effect are shown in bold. (d) PKR mutations (labeled) identified in a random screen as conferring resistance to vacK3L-H47R. PSRs are colored blue. 9 of 12 random mutations conferring resistance to vacK3L-H47R occurred at positively selected sites (cyan). The remaining three PKR mutations, which altered residues that were not identified to be under positive selection, and the PSR I405 are located in the interior of the protein and cannot be seen.

Figure 5. Effects of PKR variants on sensitivity to pseudosubstrate inhibition in HeLa cells

(a) Human HeLa PKR^kd cells were co-transfected with expression vectors for luciferase, knock-down resistant derivatives of human (h)PKR, hPKR-E379R or hPKR-A488E and control, vacK3L, vacK3L-H47R or swpvC8L. After 40 hours, cells were harvested, lysed, and samples were assayed for luciferase activity. Luciferase activity was normalized to control transfections lacking PKR inhibitors. Standard deviations are indicated for three independent transfections. Significant differences are indicated: * = p<0.005; ** = p<0.001; n.s. = not significant (p > 0.05). (b) Co-transfection of luciferase, hPKR, hPKR-A488E, mouse (m)PKR or mPKR-E451A and vacK3L or swinepox C8L. (c) Immunoblot analyses of whole cell extracts from cells transfected with the indicated expression vectors in the absence (−) or presence (+) of vacK3L. The blots were probed with antiserum specific for phosphorylated Ser51 in eIF2α (upper panel), total human eIF2α (second panel), hPKR (third panel), or mPKR (bottom panel).