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Protein kinase R reveals an evolutionary model for defeating viral mimicry.

Elde NC, Child SJ, Geballe AP, Malik HS - Nature (2008)

Bottom Line: Distinguishing self from non-self is a fundamental biological challenge.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

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.

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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)15. 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 reasons15. 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.
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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)15. 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 reasons15. 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.

Mentions: To determine if PKR might be subject to positive selection, we cloned and sequenced cDNA of PKR from a panel of 20 primates representing over 30 million years of evolutionary divergence. By considering ratios of non-synonymous (dN) and synonymous (dS) substitutions, we found evidence for ancient, episodic positive selection in primate lineages (p<0.0003; Table S1, Figure 1a). In particular, one branch in Old World monkeys was calculated to have undergone 22 non-synonymous substitutions without any synonymous changes, one of the most intense episodes of positive selection reported for any primate gene (Supplementary data). Likelihood ratio tests13 using the entire phylogeny reveal that 17% of codons have evolved with an average dN/dS ratio of 3.7, strongly supporting a finding of positive selection (p<0.0001, Table S2 and S3), even after accounting for the potentially confounding effects of recombination and synonymous site variation14 (p<0.0001; Table S4 and S5). Positive selection is observed in each of the three domains of PKR: the dsRNA binding domain, the spacer region, and even the kinase domain, consistent with an extensive history of facing viral factors that directly bind PKR in these separate domains (Figure S1). Interestingly, several residues in the kinase domain, which make direct contacts with eIF2α15, are among the fastest evolving residues in PKR (Figure 1b and S1), suggesting that selective pressure to evade eIF2α mimics may have driven changes in these residues.


Protein kinase R reveals an evolutionary model for defeating viral mimicry.

Elde NC, Child SJ, Geballe AP, Malik HS - Nature (2008)

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)15. 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 reasons15. 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.
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Related In: Results  -  Collection

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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)15. 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 reasons15. 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.
Mentions: To determine if PKR might be subject to positive selection, we cloned and sequenced cDNA of PKR from a panel of 20 primates representing over 30 million years of evolutionary divergence. By considering ratios of non-synonymous (dN) and synonymous (dS) substitutions, we found evidence for ancient, episodic positive selection in primate lineages (p<0.0003; Table S1, Figure 1a). In particular, one branch in Old World monkeys was calculated to have undergone 22 non-synonymous substitutions without any synonymous changes, one of the most intense episodes of positive selection reported for any primate gene (Supplementary data). Likelihood ratio tests13 using the entire phylogeny reveal that 17% of codons have evolved with an average dN/dS ratio of 3.7, strongly supporting a finding of positive selection (p<0.0001, Table S2 and S3), even after accounting for the potentially confounding effects of recombination and synonymous site variation14 (p<0.0001; Table S4 and S5). Positive selection is observed in each of the three domains of PKR: the dsRNA binding domain, the spacer region, and even the kinase domain, consistent with an extensive history of facing viral factors that directly bind PKR in these separate domains (Figure S1). Interestingly, several residues in the kinase domain, which make direct contacts with eIF2α15, are among the fastest evolving residues in PKR (Figure 1b and S1), suggesting that selective pressure to evade eIF2α mimics may have driven changes in these residues.

Bottom Line: Distinguishing self from non-self is a fundamental biological challenge.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

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.

Show MeSH
Related in: MedlinePlus