p-pkr(t446  (Absolute Biotech Inc)

Journal: mBio

Article Title: PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling

doi: 10.1128/mbio.00854-22

Figure Lengend Snippet: PKR binds to stem-loop domains (SLDs) 5–6 in the HRV2 IRES as a dimer, resulting in autophosphorylation of the kinase activation loop (see Fig. S2 and for extended data). (A) Schematic representation of the HRV2 IRES SLD structure with an approximate footprint for eukaryotic initiation factor (eIF) 4G:eIF4A . (B) Binding of PKR, ADAR1, PCBP2, and DHX9 to different SLD fragments as indicated. Cytosolic fractions of A375 cells, “plain” or infected with PVSRIPO (MOI 10, 48 h), were incubated with biotinylated RNA fragments (30 min) prior to pull down with Streptavidin beads. Bound proteins were analyzed by immunoblot (see Fig. S2 for corresponding input blots). (C) PKR and DHX9 binding with biotinylated wild-type (WT) SLD 5 and three distinct SLD 5 variants carrying mutations (Mut 1–3) disrupting base-pairing structure as shown. (D) C16 prevented PKR activation, evident as autophosphorylation of T446 in the kinase activation loop, upon binding to biotinylated SLD 1–3, SLD 5–6, or poly(I·C). Methylcrotonyl-coa carboxylase subunit 1 (MCCC1) nonspecifically associates with streptavidin beads (in the absence of biotinylated RNA bait) and was used as a control to ensure equal loading. The assay was performed twice, and representative results are shown. Figure S3 shows the results of a repeat in vitro phosphorylation assay. (E and F) PKR competes with eIF4G for SLD 5–6 binding. The in vitro binding assay was performed as described (see Materials and Methods), keeping constant the concentrations of recombinant GST-eIF4G(Ct), Flag-eIF4A, and biotinylated RNA bait and varying the concentration of His-SUMO-PKR for a PKR/eIF4G ratio ranging from 0.1 to 1. Immunoblots of input and proteins after RNA pull down are shown (E). The assay was performed twice, and representative results are depicted. Relative levels of eIF4G and PRK after pull down were plotted, where maximal binding was set at 100% (F). Graphs represent the averages of 2 independent experiments. *, P < 0.05, **, P < 0.005.

Article Snippet: Primary antibodies used in this study were against eIF4G1, eIF4A, GAPDH, IRF3, p-IRF3(S396), STAT1, p-STAT1(Y701), IFNβ, IFIT1, PCBP2, PKR, ADAR, Dicer, IFI16, PACT, MDA5, TBK1, p-TBK1(S172), eIF2a, p-eIF2α(S51) (all Cell Signaling Technology), DHX9, LGP2, IFIT5 (all Proteintech), HelZ2, MCCC1 (ThermoFisher), DHX30 (Novus), NF90 (BD Biosciences), α-tubulin (Sigma-Aldrich), and p-PKR(T446) (LSBio).

Techniques: Activation Assay, Binding Assay, Infection, Incubation, Western Blot, In Vitro, Phosphorylation Assay, Recombinant, Concentration Assay