Fig 2: Functional assessment of RPIA phosphoresidues.(A) A ribbon model of the predicted interaction between RPIA (cerulean) and the kinase domain of PKR (green), with the bound ATP in stick form and magnesium ions as spheres. The substrate-interacting element of PKR (the αG-helix) engages RPIA in an orientation in which a loop between the α3-helix and β4-sheet on the isomerase projects into the catalytic pocket of the kinase. (B) Spectrometric measures of the activity of the indicated RPIA constructs. Preparations of the constructs are visualized by Coomassie staining of the SDS-PAGE separated proteins (inset). Isomerase activity is compared as a t-statistic of the slopes of the trend lines of the data (n = 3). The estimated goodness-of-fit (R2) of the WT RPIA data is shown. (C–E) Plots demonstrating the impact of RPIA phosphoresidues by measures of the growth of E. coli expressing WT or kinase-dead (K296R) PKR alone or in conjunction with the indicated constructs of (C) human RPIA and (D) E. coli RpiA untreated or induced (+IPTG) at 37 °C with shaking for 24 h (n = 3). (E) Plots demonstrating that the carboxyl (C) but not the amino (N)-terminus of the isomerase relieves PKR-dependent suppression of bacterial growth after 34 h of culture (n = 3). The data show the x̅ ± σ of independent experiments. P values were calculated by unpaired student’s t-test of independent experiments. Figures are derived from Source Data file 7. Source data are available online for this figure.

Fig 3: P58IPK cosedimented with 40S ribosomal subunit in the presence of NP.A. Ribosomal subunits sucrose density gradient fractionation. Absorbance of sucrose density gradient fractions at 260 nm was plotted verses fraction number. B. HEK293T cells were transfected with plasmids expressing EGFP, NP or NPΔ151–175 mutant. The cell lysates were fractionated on sucrose density gradient to generate the ribosomal subunit and polysomes sedimentation diagram, as shown in panel A. Panel B shows the western blot analysis of 40S and 60S ribosomal subunit associated proteins based on the plot diagram in panel A. Selected fractions were mixed with equal volume of 2x SDS sample buffer, boiled and subjected to SDS-PAGE. Input represents the cleared cell lysate. RPS19, RPL4, GFP, PKR, and P58IPK were probed with its specific antibodies. NP and NPΔ 151–175 were probed with anti-Myc tag antibody.

Fig 4: dsRNA-induced pathways during SARS-CoV-2 WA1 and VOCs infections. (A) Primary nasal cultures were infected at MOI = 0.1. Cells were lysed at 96 hpi, protein extracted, and analyzed by western blot for p-PKR, PKR, p-eIF2α, and eIF2α. The controls, GAPDH and SARS-CoV-2 nucleocapsid (N), are the same as Fig. 2A. (B) A549ACE2 cells were infected at MOI = 0.1 for 72 hpi. Cells were lysed, protein extracted, and analyzed by western blot for p-PKR, PKR, p-eIF2α, eIF2α, N, and GAPDH. (A and B) Images are representative of western blots from three independent infections. (C) Infections were performed in A549ACE2 cells at MOI = 0.1. At 48 hpi (SARS-CoV-2 strains) and 24 hpi (SINV), cells were lysed, RNA extracted, and analyzed on a Bioanalyzer. Arrows indicate bands of degraded RNAs. Image is representative of two independent experiments.

Fig 5: 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.