Viral dsRNA inhibitors prevent self-association and autophosphorylation of PKR

Abstract

Host response to viral RNA genomes and replication products represents an effective strategy to combat viral invasion. PKR is a Ser/Thr protein kinase that binds to double-stranded (ds)RNA, autophosphorylates its kinase domain, and subsequently phosphorylates eukaryotic initiation factor 2alpha (eIF2alpha). This results in attenuation of protein translation, preventing synthesis of necessary viral proteins. In certain DNA viruses, PKR function can be evaded by transcription of highly structured virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We probe here the mechanism of PKR inhibition by two viral inhibitor RNAs, EBER(I) (from Epstein-Barr) and VA(I) (from human adenovirus). Native gel shift mobility assays and isothermal titration calorimetry experiments confirmed that the RNA-binding domains of PKR are sufficient and necessary for the interaction with dsRNA inhibitors. Both EBER(I) and VA(I) are effective inhibitors of PKR activation by preventing trans-autophosphorylation between two PKR molecules. The RNA inhibitors prevent self-association of PKR molecules, providing a mechanistic basis for kinase inhibition. A variety of approaches indicated that dsRNA inhibitors remain associated with PKR under activating conditions, as opposed to activator dsRNA molecules that dissociate due to reduced affinity for the phosphorylated form of PKR. Finally, we show using a HeLa cell extract system that inhibitors of PKR result in translational recovery by the protein synthesis machinery. These data indicate that inhibitory dsRNAs bind preferentially to the latent, dephosphorylated form of PKR and prevent dimerization that is required for trans-autophosphorylation.

Figure 1

Sequences and secondary structures of viral dsRNAs. Secondary structures of (A) adenovirus VA_I inhibitor , (B) Epstein-Barr virus EBER_I inhibitor , (C) the apical stem truncation of VA_I (VA_I-AS), and (D) HIV TAR dsRNA .

Figure 2

dsRBDs of PKR are sufficient and required for interaction with inhibitory dsRNAs. (A) Domain organization of PKR. N-terminal dsRBDs, C-terminal kinase domain, and the interdomain linker are shown. Critical autophosphorylation sites (T446, T451) in the kinase domain are indicated. (B) Native gel mobility shift assay for PKR derivatives (600 nM) binding to VA_I (200 nM). (C) Summary of dissociation constants (μM) at 30 °C for titration of dsRNA (10 μM, sample cell) with PKR derivatives added in trans (150 μM, syringe). Thermodynamic parameters are included in the supplemental materials.

Figure 3

Inhibitors prevent latent PKR from trans-phosphorylation. (A) Inhibition of PKR autophosphorylation in the presence of HIV-TAR activator. Either EDTA, VA_I, or EBER_I was added to a reaction mixture containing PKR, TAR, ATP and MgCl₂ at various time points as indicated. The reaction was then allowed to proceed for the complete 2 hours. Each time point was performed in triplicate, resolved by SDS-PAGE, and quantitated for ³²P incorporation by autoradiography. (B) Trans-autophosphorylation assays in which PKR^P was used to phosphorylate PKR in the presence of increasing amounts of inhibitor (VA_I or EBER_I). Reaction mixtures were incubated in the presence of [γ-³²P]ATP at 30°C for 15 minutes, and quenched by addition of EDTA. SDS-PAGE separation followed by autoradiography is shown.

Figure 4

Inhibitors of PKR prevent its self-association (A) Molecular weight of PKR-VA_I complexes (5 μM) as determined by DLS without incubation at 30 °C. (B) Concentration-dependent dimerization of PKR was examined by determining the molecular weight at the specified concentration of PKR (solid black line, squares), PKR-VA_I (solid black line, circles), PKR-EBER_I (dashed grey line, crosses), PKR-VA_I-AS (solid black line, diamonds), or PKR-TAR (dashed grey line, triangles). Each data point was repeated in triplicate and error bars reflect standard deviation associated with measurements. (C) Superdex 200 HiLoad 26/60 size exclusion chromatography elution profiles of complexes at 5 μM (bottom) and 80 μM (top).

Figure 5

Inhibitory dsRNAs remain associated with PKR under activating conditions (A) Native gel mobility-shift for VA_I, EBER_I, or VA_I-AS (200 nM) binding to PKR (400 nM) in reactions containing ATP (1 mM) and MgCl₂ (2 mM) in certain cases. Samples were incubated at 30 °C for 90 minutes, resolved on 5% TBE gels, and visualized by SybrGreenII staining. (B) PKR-dsRNA complexes were pre-assembled (300 nM), and incubated at 30 °C for 90 minutes in the presence or absence of ATP (1 mM) and MgCl₂ (1 mM). RNA release was quantified by resolving reaction components on native 5% TBE gels and dsRNA staining by SybrGreenII. Each data point represents a triplicate measurement. (C) Time-dependent molecular weight determination PKR-dsRNA complexes. Equimolar complexes of PKR and dsRNA (2 μM) were incubated in the cuvette at 30°C for the time specified prior to acquisition. ATP (1 mM) and MgCl₂ (1 mM) were included in some cases. Each data point was repeated in triplicate.

Figure 6

Inhibitors of PKR result in translational recovery. (A) HeLa S10 cell lysates containing the protein translation machinery, exogenous PKR (10 nM), and 5'-capped luciferase mRNA (50 nM) are incubated for 1 hour at 30 °C in the presence (grey) or absence (black) of HCV-IRES activator dsRNA (50 nM) and increasing amounts of VA_I inhibitor (0−2 μM). Measurements were performed in triplicate and standardized relative to maximum Luciferase activity. (B) Western blot probed with an anti-(P-Ser51)-eIF2a antibody (ab4837−50, abcam) for selected reactions shown in (A).

Figure 7

Model for the inhibition of PKR kinase activity. Model summarizing the framework for the inhibition of PKR; dsRBDs (R), kinase domain (K) and interdomain linker (L). Upon binding of activator dsRNA to the dsRBDs of PKR in the latent form, enhanced bimolecular interaction between two PKR molecules is observed. In this conformation, autophosphorylation occurs, leading to RNA release and activation competency of PKR. Subsequently, activated PKR may feed back to trans-phosphorylate remaining latent PKR. Addition of inhibitory dsRNA leads to interaction with the latent form of PKR; binding prevents dimerization and prevents PKR from acting as a substrate for trans-autophosphorylation. Structured stem-loops not required for interaction with the dsRBDs may be responsible for mediating inhibition.