Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR

The antiviral protein kinase R (PKR) is an important restriction factor, which poxviruses must overcome to productively infect host cells. Many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus, to inhibit PKR. Although the interaction between PKR and eIF2alpha is highly conserved, some K3 orthologs were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that the amino acid combinations needed to mediate improved or diminished inhibition of PKR varied between different species. The data presented here show that even closely related species can display drastically different sensitivities to orthopoxvirus PKR inhibitors, and that orthologs from closely related poxviruses can show strong differences in their function. Furthermore, there is likely to be a limited number of possible mutations that disrupt K3-interactions but still maintain PKR/eIF2alpha interactions. Thus, by chance some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral host antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined.