A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

Abstract

During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α.

Species‐specific inhibition of antiviral protein kinase R by capripoxviruses and vaccinia virus

“…The effects of differential PKR inhibition have so far been addressed in yeast assays and cultured cells, in which yeast growth, eIF2␣, and PKR phosphorylation, mRNA translation, or virus replication have been used as readouts for PKR inhibition. [9][10][11]19,41 Currently, we have very limited information about the effects of differential PKR inhibition from in vivo infection models. It seems clear that no or ineffective PKR inhibition would result in abortive infection in vivo.…”

“…It has been shown that positively selected amino acid residues contribute directly to PKR sensitivity to inhibitors from poxviruses and herpesviruses. [9][10][11] Most poxviruses encode two PKR inhibitors, which are called K3 (encoded by K3L) and E3 (encoded by E3L) in VACV, the prototypic poxvirus. 3 K3 is an eIF2␣ homolog and is thought to act as a pseudosubstrate inhibitor by binding to activated, phosphorylated PKR to prevent its interaction with eIF2␣.…”

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

“…Human CMV TRS1 only inhibited human PKR but not PKR from old world monkeys while the TRS1 ortholog from African green monkey CMV inhibited AGM but not human PKR. Intriguingly, mutation of a single amino acid in human PKR (position 489), which determines susceptibility to human or AGM TRS1, is also involved in the weak sensitivity of human PKR to VACV K3 inhibition, highlighting that inhibitors from different pathogens can be affected by mutations in important antiviral proteins [8,10].…”

“…HeLa (human, ATCC #CCL-2), HeLa PKR-knock-down( kd ), kindly provided by Dr. Charles Samuel [44], HeLa PKR-knock-out, kindly provided by Dr. Adam Geballe [10], RK13+E3L+K3L cells (rabbit) [45], tert-GF (tert immortalized gibbon fibroblasts, kindly supplemented with 5% fetal bovine serum or 10% horse serum (BT cells) and 100 IU/ml penicillin/streptomycin (Gibco). HeLa PKR kd cells were maintained in medium containing 1 µg/ml puromycin (Sigma).…”

“…These conflicting host and viral proteins exert strong selective pressure on each other, driving rapid, successive changes to the coding sequence of each protein to either disrupt or re-engage the interaction. Signatures of positive selection resulting from this evolutionary "arms race" have been identified across the PKR coding sequence, and these positively selected residues have been shown to be determinants of differential susceptibility to both poxvirus and herpesvirus PKR antagonists [8][9][10].…”