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. 2017 Feb 14;91(5):e01574-16.
doi: 10.1128/JVI.01574-16. Print 2017 Mar 1.

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

Heather A Vincent  1 , Benjamin Ziehr  1 , Nathaniel J Moorman  2
Affiliations

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1

Heather A Vincent et al. J Virol. .

Abstract

Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2α. Consistent with previous in vitro studies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2α binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2α contact site in PKR binding, pTRS1 bound an additional eIF2α kinase, heme-regulated inhibitor (HRI), and inhibited eIF2α phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2α binding site and blocking PKR kinase activity.IMPORTANCE The antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease.

Keywords: HCMV; PKR; eIF2α; gene expression; human cytomegalovirus; human herpesvirus; innate immunity; mRNA translation; protein kinase R; protein synthesis.

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Figures

FIG 1
FIG 1
Domains of pTRS1 necessary for PKR binding and inhibition. (A) Lysates from HEK293T cells transfected with either a GFP or pTRS1 expression vector were treated with poly(I·C) to induce PKR activation. Levels of phosphorylated PKR (T446) were measured by Western blotting. (B) Schematic showing location of pTRS1 functional domains and pTRS1 truncation mutants (RBD, dsRNA binding domain; PBD, PKR binding domain). Areas shaded gray are conserved between pTRS1 and pIRS1. (C) HEK293T cells were transfected with GFP, full-length TRS1, or the indicated TRS1 mutants. Lysates were treated with poly(I·C), and PKR phosphorylation (T446) was measured by Western blotting. (D) HEK293T cells were cotransfected with Myc-tagged kinase-dead PKR (K296R) and either full-length TRS1 or the indicated TRS1 mutants. Cell lysates were immunoprecipitated with anti-Myc (PKR) or IgG (specificity control) antibody. The presence of pTRS1 in the immune complexes was determined by Western blotting. Representative results from one of at least three independent experiments are shown for each panel.
FIG 2
FIG 2
PKR binding by full-length pTRS1 is necessary for inhibition of PKR activation. (A) Lysates from HEK293T cells transfected with full-length TRS1 (WT) or the indicated TRS1 mutants. pTRS1 1-550 (1-550) expresses only the first 550 amino acids of pTRS1. pTRS1triple (Triple) contains mutations that disrupt dsRNA binding. pTRS1 1-550triple (1-550 Triple) expresses the 1-550 truncation mutant containing mutations in the dsRNA binding domain. Lysates were incubated with poly(I·C) to activate PKR. PKR phosphorylation (T446) was examined by Western blotting. (B) Same as panel A, except with different TRS1 mutants. pTRS1mut1 (Mut1) contains mutations that disrupt PKR binding. (C) HEK293T cells were transfected with Myc-tagged kinase-dead PKR (K296R) together with full-length TRS1 or the indicated TRS1 mutants. pTRS1mut1.triple (Mut1-triple) contains point mutations in both the PKR and dsRNA binding domains. Cell lysates were immunoprecipitated with anti-Myc (PKR) or IgG (specificity control) antibody. The presence of pTRS1 in the immune complexes was determined by Western blotting. (D) Same as panel C, except that lysates were treated with micrococcal nuclease prior to immunoprecipitation. (E) MRC-5 fibroblasts were infected with HCMV at a multiplicity of infection (MOI) of 3 for 24 h. The presence of PKR in pTRS1 or IE1 specific immune complexes was determined by Western blotting. Representative results from one of at least three independent experiments are shown for each panel.
FIG 3
FIG 3
pTRS1 does not prevent PKR dimerization. HEK293T cells expressing pTRS1 from an inducible promoter (293T-pTRS1i) were cotransfected with Flag- and Myc-tagged PKR K296R expression vectors. pTRS1 expression was induced with doxycycline treatment. Cell lysates were treated with poly(I·C), and PKR was immunoprecipitated with anti-Myc antibody. The interaction of Flag- and Myc-tagged PKR was examined by Western blotting. Representative results from one of at least three independent experiments are shown.
FIG 4
FIG 4
pTRS1 binds and inhibits a constitutively active PKR kinase domain. (A) HEK293T cells were cotransfected with a doxycycline-inducible GST-tagged PKR kinase domain (GST-KD) expression vector and an expression vector encoding either pTRS1 or pTRS1mut1 (Mut1). GST-KD expression was induced with doxycycline treatment. Glutathione resin was used to capture the GST-KD, and the copurification of pTRS1 or pTRS1mut1 was examined by Western blotting. (B) Cells were transfected as in panel A, with the addition of cells cotransfected with a GFP expression vector and the GST-KD expression vector. GST-KD expression was induced with doxycycline treatment, and levels of GST-KD autophosphorylation (T446) were determined by Western blotting. Representative results from one of at least three independent experiments are shown for each panel.
FIG 5
FIG 5
pTRS1 interacts with the PKR kinase domain to prevent PKR autophosphorylation. (A) PyMOL image of the PKR kinase domain and eIF2a (PDB code 2A1A). The PKR aG helix is represented in cyan. Amino acids required for eIF2a binding are shown in magenta, while mutations that confer resistance to inhibition by K3L are shown in blue. (B) 293T-pTRS1i cells were transfected with Myc-tagged PKR K296R or the indicated Myc-tagged PKR mutants. pTRS1 expression was induced with doxycycline treatment before lysates were immunoprecipitated with anti-Myc (PKR) or IgG (specificity control) antibody. The presence of pTRS1 in the immune complexes was determined by Western blotting. (C) PKR-deficient HeLa cells (PKR KO) were transfected with the indicated PKR mutants together with vectors expressing either GFP or TRS1. Cells were transfected with poly(I·C) to activate PKR and PKR autophosphorylation (T446) was measured by Western blotting. (D and E) Same as panels B and C, except that cells were transfected with the indicated PKR mutants. Representative results from one of at least three independent experiments are shown for each panel.
FIG 6
FIG 6
pTRS1 inhibits PKR kinase activity. HEK293T cells were transfected with the indicated expression vectors (PKR K296R, kinase-dead PKR; Mut1, pTRS1 containing PKR binding domain mutations). PKR was immunoprecipitated with anti-Myc antibody and resuspended in kinase buffer. The kinase reaction was performed “on bead” as described in Materials and Methods, using histone H2A peptide as a substrate. PKR kinase activity was measured using the ADP-Glo kinase assay. The amount of kinase activity is normalized to the background activity associated with nonspecific (IgG) immunoprecipitates, which was set to 1. Statistical significance was determined using an unpaired Student t test (n = 3; *, P < 0.05).
FIG 7
FIG 7
pTRS1 binds HRI and limits its activation. (A) 293T-pTRS1i cells were transfected with Myc-tagged HRI, and pTRS1 expression was induced with doxycycline. Lysates were immunoprecipitated with anti-Myc (PKR) or control antibody (αTubulin), and the presence of HRI the immune complexes was determined by Western blotting. (B) MRC-5 fibroblasts were infected with HCMV at an MOI of 3 for 72 h. The amount of HRI that was copurified with pTRS1 specific immune complexes or with beads alone (control) was determined by Western blotting. (C) PKR KO-pTRS1i cells were treated with doxycycline to induce pTRS1 expression and subsequently treated with increasing concentrations of arsenite (ARS) to activate HRI. Levels of eIF2α phosphorylation were measured by Western blotting. The ratio of phosphorylated eF2α to total eIF2α levels (P/total) was calculated by densitometry. Representative results from one of at least three independent experiments are shown for each panel.
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