Systematic screen of PKR reveals genetic variants that broadly evade divergent viral pseudosubstrate inhibitors

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 by phosphorylating eIF2, a component of the translation initiation machinery. PKR is targeted by many viral antagonists, including pseudosubstrate inhibitors encoded by poxviruses and ranaviruses that mimic eIF2 and inhibit PKR activity. We previously found that the eIF2-binding surface of human PKR is highly malleable against the vaccinia virus pseudosubstrate inhibitor K3. Here, we extend that work using our PKR library of 426 SNP-accessible variants against four additional viral pseudosubstrate inhibitors with increasing sequence diversity: K3 orthologs from variola virus, tanapox virus, and myxoma virus, as well as the independently derived eIF2 mimic vIF2 from Rana catesbeiana virus Z. We find that resistance-conferring variants are readily accessible against all inhibitors tested and are often shared across phylogenetically diverse poxvirus K3 orthologs and the independently derived ranavirus inhibitor, suggesting that PKR escape variants can exploit features common to pseudosubstrate inhibitors. Variants beneficial against multiple inhibitors clustered in alpha helices D and G of the PKR kinase domain, and many correspond to sites under positive selection across vertebrates. Inhibitor-specific effects could largely be explained by differences in contact residues between PKR and each inhibitor. Notably, no PKR variant became newly susceptible to myxoma K3, which does not naturally inhibit human PKR. Overall, we find that the eIF2-binding surface of PKR is broadly navigable against genetically diverse viral pseudosubstrate inhibitors, potentiating its evolutionary ability to combat viral inhibition without necessarily incurring new vulnerabilities.