Systematic genetic characterization of the human PKR kinase domain highlights its functional malleability to escape a viral substrate mimic
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eLife assessment
This valuable report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. A strength of the manuscript is the powerful combination of a yeast-based assay with high-throughput sequencing and its convincing experimental use to characterize large numbers of PKR variants. A minor weakness is that the scope of the screen conducted could still be extended, for example in terms of the segments of PKR included.
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Abstract
Evolutionary arms races can arise at the contact surfaces between host and viral proteins, producing dynamic spaces in which genetic variants are continually pursued. However, the sampling of genetic variation must be balanced with the need to maintain protein function. A striking case is given by protein kinase R (PKR), a member of the mammalian innate immune system. PKR detects viral replication within the host cell and halts protein synthesis to prevent viral replication by phosphorylating eIF2α, a component of the translation initiation machinery. PKR is targeted by many viral antagonists, including poxvirus pseudosubstrate antagonists that inhibit PKR by interacting with the same binding surface as eIF2α. Remarkably, PKR has several rapidly evolving residues at this interface, suggesting it is engaging in an evolutionary arms race, despite the surface’s critical role in phosphorylating eIF2α. To systematically explore the evolutionary opportunities available at this dynamic interface, we generated and characterized a library of 426 SNP-accessible nonsynonymous variants of human PKR for their ability to escape inhibition by the model pseudosubstrate inhibitor K3 from vaccinia virus. We identified key sites in the PKR kinase domain that harbor K3-resistant variants, as well as critical sites where variation leads to loss of function. We find K3-resistant variants are readily available throughout the interface and are enriched at sites under positive selection. Moreover, variants beneficial against K3 were also beneficial against an enhanced variant of K3, indicating resilience to viral adaptation. Overall, we find that the eIF2α-binding surface of PKR is highly malleable, potentiating its evolutionary ability to combat viral inhibition.
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eLife assessment
This valuable report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. A strength of the manuscript is the powerful combination of a yeast-based assay with high-throughput sequencing and its convincing experimental use to characterize large numbers of PKR variants. A minor weakness is that the scope of the screen conducted could still be extended, for example in terms of the segments of PKR included.
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Reviewer #1 (Public Review):
Summary:
The report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. Repressive binding of K3 to the kinase prevents phosphorylation of its recognised substrate, EIF2α (the α subunit of the Eukaryotic Initiation Factor 2). The interaction of K3 is probed by saturation mutation within four regions of PKR chosen by modelling the molecules' interaction. They identify K3-resistant PKR variants that recognise that the K3/EIF2α-binding surface of the kinase is malleable. This is reasonably interpreted as indicating the potential adaptability of this antiviral protein to combat viral virulence factors.
Strengths:
This is a well-conducted study that probes the versatility of the antiviral response to escape a viral inhibitor. The experimentation is …
Reviewer #1 (Public Review):
Summary:
The report describes the control of the activity of the RNA-activated protein kinase, PKR, by the Vaccinia virus K3 protein. Repressive binding of K3 to the kinase prevents phosphorylation of its recognised substrate, EIF2α (the α subunit of the Eukaryotic Initiation Factor 2). The interaction of K3 is probed by saturation mutation within four regions of PKR chosen by modelling the molecules' interaction. They identify K3-resistant PKR variants that recognise that the K3/EIF2α-binding surface of the kinase is malleable. This is reasonably interpreted as indicating the potential adaptability of this antiviral protein to combat viral virulence factors.
Strengths:
This is a well-conducted study that probes the versatility of the antiviral response to escape a viral inhibitor. The experimentation is very diligent, generating and screening a large number of variants to recognise the malleability of residues at the interface between PKR and K3.
Weaknesses:
These are minor. The protein interaction between PKR and K3 has been previously well-explored through phylogenetic and functional analyses and molecular dynamics studies, as well as with more limited site-directed mutational studies using the same experimental assays. Accordingly, these findings largely reinforce what had been established rather than making major discoveries.
There are some presumptions:
It isn't established that the different PKR constructs are expressed equivalently so there is the contingency that this could account for some of the functional differences.
Details about the confirmation of PKR used to model the interaction aren't given so it isn't clear how accurately the model captures the active kinase state. This is important for the interaction with K3/EIF2α.
Not all regions identified to form the interface between PKR and K3 were assessed in the experimentation. It isn't clear why residues between positions 332-358 weren't examined, particularly as this would have made this report more complete than preceding studies of this protein interaction.
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Reviewer #2 (Public Review):
Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.
The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are …
Reviewer #2 (Public Review):
Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.
The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are enriched at sites under positive selection. They further found some evidence of PKR resilience to viral antagonist diversification. These findings highlight the remarkable adaptability of PKR in response to viral antagonism by mimicry.
Significance of the findings:
The findings are important with implications for various fields, including evolutionary biology, virus-host interfaces, genetic conflicts, and antiviral immunity.
Strength of the evidence:
Convincing methodology using state-of-the-art mutational scanning approach in an elegant and simple setup to address important challenges in virus-host molecular conflicts and protein adaptations.
Strengths:
● Systematic and Unbiased Approach:
The study's comprehensive approach to generating and characterizing a large library of PKR variants provides valuable insights into the evolutionary landscape of the PKR kinase domain. By focusing on SNP-accessible variants, the authors ensure the relevance of their findings to naturally occurring mutations.● Identification of Key Sites:
The identification of specific sites in the PKR kinase domain that confer resistance or susceptibility to a poxvirus pseudosubstrate inhibition is a significant contribution.● Evolutionary Implications:
The authors performed meticulous comparative analyses throughout the study between the functional variants from their mutagenesis screen ("prospective") and the evolutionarily-relevant past adaptations ("retrospective").● Experimental Design:
The use of a yeast-based assay to simultaneously assess PKR capacity to induce cell growth arrest and susceptibility/resistance to various VACV K3 alleles is an efficient approach. The combination of this assay with high-throughput sequencing allows for the rapid characterization of a large number of PKR variants.Areas for Improvement:
● Validation of the screen:
The results would be strengthened by validating results from the screen on a handful of candidate PKR variants, either using a similar yeast heterologous assay, or - even more powerfully - in another experimental system assaying for similar function (cell translation arrest) or protein-protein interaction.● Evolutionary Data:
Beyond residues under positive selection, the screen would allow the authors to also perform a comparative analysis with PKR residues under purifying selection. Because they are assessing one of the most conserved ancestral functions of PKR (i.e. cell translation arrest), it may also be of interest to discuss these highly conserved sites.● Mechanistic Insights:
While the study identifies key sites and residues involved in vaccinia K3 resistance, it could benefit from further investigation into the underlying molecular mechanisms. The study's reliance on a single experimental approach, deep mutational scanning, may introduce biases and limit the scope of the findings. The authors may acknowledge these limitations in the Discussion.● Viral Diversity:
The study focuses on the viral inhibitor K3 from vaccinia. Expanding the analysis to include other viral inhibitors, or exploring the effects of PKR variants on a range of viruses would strengthen and expand the study's conclusions. Would the identified VACV K3-resistant variants also be effective against other viral inhibitors (from pox or other viruses)? or in the context of infection with different viruses? Without such evidence, the authors may check the manuscript is specific about the conclusions.Overall Assessment:
The systematic approach, identification of key sites, and evolutionary implications are all notable strengths. While there is room for further investigation into the mechanistic details and broader viral diversity, the findings are robust and already provide important advancements. The manuscript is well-written and clear, and the figures are informative. Specific minor comments are further shared below.
Minor revisions addressing the areas for improvement mentioned above are recommended.
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Reviewer #3 (Public Review):
Summary:
- This study investigated how genetic variation in the human protein PKR can enable sensitivity or resistance to a viral inhibitor from the vaccinia virus called K3.
- The authors generated a collection of PKR mutants and characterized their activity in a high-throughput yeast assay to identify 1) which mutations alter PKR's intrinsic biochemical activity, 2) which mutations allow for PKR to escape from viral K3, and 3) which mutations allow for escape from a mutant version of K3 that was previously known to inhibit PKR more efficiently.
- As a result of this work, the authors generated a detailed map of residues at the PKR-K3 binding surface and the functional impacts of single mutation changes at these sites.
Strengths:
- Experiments assessed each PKR variant against three different alleles of the …
Reviewer #3 (Public Review):
Summary:
- This study investigated how genetic variation in the human protein PKR can enable sensitivity or resistance to a viral inhibitor from the vaccinia virus called K3.
- The authors generated a collection of PKR mutants and characterized their activity in a high-throughput yeast assay to identify 1) which mutations alter PKR's intrinsic biochemical activity, 2) which mutations allow for PKR to escape from viral K3, and 3) which mutations allow for escape from a mutant version of K3 that was previously known to inhibit PKR more efficiently.
- As a result of this work, the authors generated a detailed map of residues at the PKR-K3 binding surface and the functional impacts of single mutation changes at these sites.
Strengths:
- Experiments assessed each PKR variant against three different alleles of the K3 antagonist, allowing for a combinatorial view of how each PKR mutant performs in different settings.
- Nice development of a useful, high-throughput yeast assay to assess PKR activity, with highly detailed methods to facilitate open science and reproducibility.
- The authors generated a very clean, high-quality, and well-replicated dataset.
Weaknesses:
- The authors chose to focus solely on testing residues in or near the PKR-K3 predicted binding interface. As a result, there was only a moderately complex library of PKR mutants tested. The residues selected for investigation were logical, but this limited the potential for observing allosteric interactions or other less-expected results.
- For residues of interest, some kind of independent validation assay would have been useful to demonstrate that this yeast fitness-based assay is a reliable and quantitative readout of PKR activity.
- As written, the current version of the manuscript could use more context to help a general reader understand 1) what was previously known about these PKR and K3 variants, 2) what was known about how other genes involved in arms races evolve, or 3) what predictions or goals the authors had at the beginning of their experiment. As a result, this paper mostly provides a detailed catalog of variants and their effects. This will be a useful reference for those carrying out detailed, biochemical studies of PKR or K3, but any broader lessons are limited.
I felt there was a missed opportunity to connect the study's findings to outside evolutionary genetic information, beyond asking if there was overlap with PKR sites that a single previous study had identified as positively selected. For example, are there any signals of balancing selection for PKR? How much allelic diversity is there within humans, and are people typically heterozygous for PKR variants? Relatedly, although PKR variants were tested in isolation here, would the authors expect their functional impacts to be recessive or dominant, and would this alter their interpretations? On the viral diversity side, how much variation is there among K3 sequences? Is there an elevated evolutionary rate, for example, in K3 at residues that contact PKR sites that can confer resistance? None of these additions are essential, but some kind of discussion or analysis like this would help to connect the yeast-based PKR phenotypic assay presented here back to the real-world context for these genes.
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