HuPKR F489S confers resistance in the context of viral replication.

(A) Triplicate wells of HeLa PKR KO cells inducibly expressing the indicated PKR variants were treated +/- doxycycline and infected (MOI 0.1) with a panel of VacVs. At 48 hpi, viral replication was quantified by measuring β-gal activity and is reported as percent replication in doxycycline treated cells relative to replication in the same cells without induction of PKR expression (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected empty vector cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length. (C) PKR expression in lysates of mock-infected cells from (A) was evaluated by western blotting. *AgmPKR does not cross react with the antibody used.</p

Mechanisms of viral capture.

The liver appears to be the main mediator of viral vascular clearance. However, the specific mechanisms of removing virions from the circulation is distinct and virus-specific. The removal of AdV particles is mainly performed by KCs; however, some of the receptors shown to interact with AdV can also be expressed by LSECs (SR-F1 and SR-A1). In addition to SRs (SR-F1, SR-A1, and SR-A6), nAb, and CRIg also promote clearance of AdV from the bloodstream. For arthritogenic alphaviruses (CHIKV, RRV, and ONNV), clearance is mediated specifically by SR-A6 (MARCO) and KCs. However, particles that have a single point mutation to replace a lysine residue on the E2 glycoprotein (K200X for CHIKV and ONNV; K251X for RRV) evade capture. For the flaviviruses DENV and WNV, the type of virion glycosylation present affects clearance mediated by MBL. Specifically, MBL binds the high-mannose glycosylated virus particles, but not virions decorated with complex glycosylation. However, MBL is not the only mediator of DENV and WNV clearance, and it is clear another, as-yet-unknown mechanism also exists. This figure was created with BioRender.com. AdV, adenovirus; CHIKV, chikungunya virus; DENV, dengue virus; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell; MBL, mannose-binding lectin; nAb, natural antibodies; ONNV, o’nyong’nyong virus; RRV, Ross River virus; WNV, West Nile virus.</p

Documented surface-expressed pattern recognition receptors of LSECs and KCs.

Documented surface-expressed pattern recognition receptors of LSECs and KCs.</p

Host mechanisms of viral clearance.

Host mechanisms of viral clearance.</p

Macrophages of the spleen.

Splenic macrophages also participate in the capture of circulating virus particles. There are 3 major splenic macrophage populations (MMM, MZM, and RpM), and they localize to distinct regions of the spleen. These macrophage subsets can be identified by their localization and the indicated key cellular markers. While the mechanisms by which specific splenic macrophage populations mediate viral clearance are not well understood, they are critical in activating immune responses to circulating viruses. This figure was created with BioRender.com. MMM, marginal zone metallophilic macrophage; MZM, marginal zone macrophage; RpM, red pulp macrophage.</p

The liver sinusoid.

There are 2 key cell types located in the liver sinusoid that have been shown to contribute to viral vascular clearance. Although in vitro studies suggest that LSECs, which form the liver endothelium, interact with certain viruses (e.g., AdV), KCs, which are the liver’s main tissue-resident macrophages, are responsible for clearing diverse circulating viruses (e.g., CHIKV and AdV) in vivo. In addition, KCs are important in controlling pathogenesis of viruses like LCMV. This figure was created with BioRender.com. AdV, adenovirus; CHIKV, chikungunya virus; HA, hepatic artery; IFN, interferon; KC, Kupffer cell; LCMV, lymphocytic choriomeningitis virus; LSEC, liver sinusoidal endothelial cell; PRR, pathogen recognition receptor; PV, portal vein; VACV, vaccinia virus.</p

The F489S mutation eliminates HCMV_TRS1 binding to PKR.

<p>HeLa PKR KO cells were co-transfected with WT HuPKR or HuPKR F489S and either His-tagged HCMV_TRS1, AgmCMV_TRS1, SmCMV_TRS1 or EGFP. At 48h post transfection, lysates were prepared and incubated with nickel-agarose beads. Cell lysates and bound proteins were analyzed by western blotting, probing for His-tagged TRS1 proteins and for PKR. Data are representative of three independent experiments.</p

Species-specific differences in primate CMV TRS1 PKR antagonism.

<p>(A) HeLa PKR KO, HeLa (human), or BSC40 (Agm) cells were mock infected or infected (MOI 0.1) with WT VacV, VacVΔE3L, or VacVΔE3L recombinants containing HCMV_TRS1, AgmCMV_TRS1, RhCMV_TRS1, or SmCMV_TRS1. At 48 hpi, viral replication was quantified by measuring β-gal activity (mean ± s.d., n = 3). Data are representative of three independent experiments. (B) His-tagged TRS1 constructs were detected in lysates of the infected HeLa PKR KO cells from (A) by western blotting. TRS1 size variation is expected based on differences in coding length.</p

Species-specific differences in primate CMV TRS1 PKR antagonism map to a single amino acid.

<p>(A) Schematic representation of the SEAP assay. Transfection of PKR leads to decreased activity of a co-transfected reporter construct expressing SEAP. This PKR-driven repression can be counteracted by co-transfection of a functional TRS1 antagonist, resulting in a rescue of SEAP activity. (B) The SEAP assay recapitulates species-specific differences in HuPKR antagonism by TRS1 alleles. HeLa PKR KO cells were co-transfected with a SEAP reporter plasmid along with either a control vector or HuPKR and the indicated TRS1 alleles or a vector control. At 48 h post-transfection, SEAP activity in the medium was measured (mean ± s.d., n = 2). Data are representative of three independent experiments. (C) A single amino acid change, F489S, confers resistance to HCMV_TRS1. Point mutants were generated in HuPKR to introduce the six AgmPKR-specific residues that differ between HuPKR and AgmPKR within the region spanning codons 475 to 520, shown in the alignment. The ability of the point mutants to antagonize HuPKR was evaluated as described in (B) (mean ± s.d., n = 2). Data are representative of three independent experiments.</p

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

<div><p>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 HCMV_TRS1. 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.</p></div