Magnesium-dependent interaction of PKR with adenovirus VAI

Abstract

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Activated PKR phosphorylates the α-subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis in virus-infected cells. Viruses have evolved diverse PKR-inhibitory strategies to evade the antiviral response. Adenovirus encodes virus-associated RNA I (VAI), a highly structured RNA inhibitor that binds PKR but fails to activate. We have characterized the stoichiometry and affinity of PKR binding to define the mechanism of PKR inhibition by VAI. Sedimentation velocity and isothermal titration calorimetry measurements indicate that PKR interactions with VAI are modulated by Mg(2+). Two PKR monomers bind in the absence of Mg(2+), but a single monomer binds in the presence of divalent ion. Known RNA activators of PKR are capable of binding multiple PKR monomers to allow the kinase domains to come into close proximity and thus enhance dimerization. We propose that VAI acts as an inhibitor of PKR because it binds and sequesters a single PKR in the presence of divalent cation.

Figure 1

Secondary structures of RNAs used in this study. A, Wild type VAI; B, ΔTS mutant lacking the terminal stem. The secondary structures are based on enzymatic structure probing measurements. RNAs were prepared by in vitro transcription from plasmid templates as previously described; and were purified on preparative denaturing gels followed by electroelution. The RNAs were annealed by heating to 90°C followed by snap cooling.

Figure 2

Sedimentation velocity analysis of PKR binding to VAI. A) Normalized g(s*) distributions of 0.4 µM VAI (black), VAI + 1 eq. PKR (green), VAI + 2 eq. PKR (blue) and VAI + 6 eq. PKR (red). Distributions were calculated using DCDT+. B) Global analysis of difference curves. The data were subtracted in pairs to remove systematic noise and the four data sets at the indicated ratios of PKR: VAI were fit to 2:1 binding model using SEDANAL. Fitted parameters were the sedimentation coefficients of the complexes, the VAI loading concentrations and the dissociation constants The top panels show the data (points) and fit (solid lines) and the bottom panels show the residuals (points). The best fit parameters are shown in Table 1. For clarity, only every 2^nd difference curve is shown. Samples were prepared in a buffer consisting of 20 mM HEPES pH 7.5, 200 mM NaCl, 0.1 mM EDTA and 0.01 mM TCEP (AU 200) and loaded into two channel aluminum-epon double-sector cells equipped with quartz windows. Data were collected using absorbance optics in a Beckman-Coulter XL-I analytical ultracentrifuge. Conditions: rotor speed, 35,000 RPM; temperature, 20°C; wavelength, 260 nm.

Figure 3

ITC analysis of PKR binding to VAI. Measurements were performing using a VP-ITC (Microcal, Inc.) at 20°C. Both RNA and protein were exchanged in AU 200 buffer by exhaustive dialysis. The calorimeter cell contained 4 µM VAI and the syringe contained 75 µM PKR. A single 2-µL injection was performed followed by 29 10-µL injections. The top panel shows the background-corrected ITC data and the bottom panel shows the integrated data (points) and fit of the data to a model of two independent binding sites (solid line) performed using ORIGIN (Microcal, Inc.) The best fit parameters are shown in Table 2.

Figure 4

Inhibition of dsRNA activation of PKR by VAI and ΔTS. PKR autophosphorylation assays were performed as previously described; in AU 200 + 5 mM MgCl₂. 100 nM PKR and variable concentrations of VAI were pre-incubated for 10 minutes at 30°C, followed by addition of 10 ug/ml poly(rI:rC). After 20 minutes, 0.4 mM ATP containing 4µCi γ-³²P[ATP] was added and the reaction was incubated for another 40 minutes before quenching with sample loading buffer. PKR autophosphorylation was quantified by phosphorimager analysis and was referenced to samples containing no inhibitor. Each point represents the average of three measurements and the error bars indicate the estimated standard error.