Systematic characterization of the local evolutionary space available to human PKR and vaccinia virus K3

The interfaces between host and viral proteins can be dynamic spaces in which genetic variants are continually pursued, giving rise to evolutionary arms races. One such scenario is found between the mammalian innate immunity protein PKR (protein kinase R) and the poxvirus antagonist K3. Once activated, PKR phosphorylates the natural substrate eIF2, which halts protein synthesis within the cell and prevents viral replication. K3 acts as a pseudosubstrate antagonist against PKR by directly antagonizing this halt in protein synthesis, enabling poxviruses to replicate in the cell. Exploring the impact of genetic variants in both PKR and K3 is necessary not only to highlight residues of evolutionary constraint and opportunity but also to elucidate the mechanism by which human PKR is able to subvert a rapidly evolving viral antagonist. To systematically explore this dynamic interface, we have generated a combinatorial library of PKR and K3 missense variants to be co-expressed and characterized in a high-throughput yeast selection assay. This assay allows us to characterize hundreds of thousands of unique PKR-K3 genetic combinations in a single pooled experiment. Our results highlight specific missense variants available to PKR that subvert the K3 antagonist. We find that improved PKR variants are readily available at sites under positive selection, with limited opportunity at sites interfacing with K3 and eIF2. Additionally, we find many variants that improve and disable K3 antagonism, suggesting a pliable interface. We reason that this approach can be leveraged to explore the evolutionary plasticity of many other host-virus interfaces.