Poxvirus K3 Orthologs Regulate NF-kB-Dependent Inflammatory Responses by Targeting the PKR–eIF2a Axis in Multiple Species

Protein kinase R (PKR) is an interferon (IFN)-inducible antiviral factor, which inhibits general mRNA translation by phosphorylating the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2). PKR also regulates NF-kB activity after virus infection; however, PKR-mediated NF-kB responses across species and their modulation by viral inhibitors remain largely unexplored. We have previously shown that the myxoma virus PKR inhibitor M156 inhibited PKR from its natural brush rabbit host better than PKR from European rabbits, and that this correlated with distinct NF-kB responses. This study aimed to elucidate the impact of 17 mammalian PKR orthologs on general translation, stress-responsive translation, and NF-kB activity using reporter gene assays and measuring NF-kB target gene induction. We found that all 17 tested PKR orthologs showed strong inhibition of general translation, strong induction of stress-responsive translation, and strong induction of NF-kB target genes TNFa and IL-6 transcripts. These NF-kB responses were only inhibited by poxviral K3 orthologs that robustly inhibited PKR activation. We also compared the effects of human and European rabbit PKR on replication of a myxoma virus strain lacking PKR inhibitors in congenic human and rabbit cells and found comparable strong inhibition of virus replication and both PKR-dependent and independent induction of TNFa and IL-6 transcripts. To elucidate the molecular basis for differential sensitivity of rabbit PKRs to M156, we found that swapping four amino acid residues between the rabbit PKR orthologs in helix aG reversed sensitivities to M156 inhibition, and that only the intermediate inhibition of wild type and mutated PKR resulted in strong NF-kB activity. Collectively, our data show that the tested PKR orthologs exhibit conserved dual antiviral and inflammatory regulatory roles, which can be antagonized by poxviral K3 orthologs that exploit eIF2a mimicry to modulate the PKR-NF-kB axis.