Inhibition of PKR by Adenovirus-Associated RNA I

Protein kinase R (PKR) is a component of the innnate immunity pathway that is activated by dsRNA to undergo dimerization and autophosphorylation. Adenovirus virus-associated RNA I (VA I) is a short, non-coding transcript that functions to inhibit the activity of PKR in the host cell by acting as an RNA decoy. VA I contains three domains: an apical stem-loop, a central domain, and a terminal stem. Previous work suggests that PKR binding is localized to the apical stem and central domain regions. We have characterized the PKR binding stoichiometry and affinity using sedimentation velocity analytical ultracentrifugation and isothermal titration calorimetry. Although two PKR molecules clearly bind to VA I in the absence of divalent ion, only one PKR binds in the presence of Mg2+ and the binding affinity is reduced by about 20-fold. In contrast, PKR binding to regular dsRNAs is not strongly affected by divalent ion. Thus, Mg2+ may be required for VA I to fold. Interestingly, we do not detect large structural changes in the RNA by small angle X-ray scattering upon addition of Mg2+. Removal of the VA I terminal stem does not affect PKR binding affinity or inhibition. PKR binds more strongly to the highly- structured, viral RNA relative to a simple dsRNA with a length comparable to the apical stem or to the isolated apical stem itself, indicating that PKR specifically recognizes the central domain. Our data indicate that VA I inhibits PKR because it binds tightly but does not foster PKR dimerization in the presence of Mg2+.