Domain interactions in adenovirus VAI RNA mediate high-affinity PKR binding.

Protein kinase R (PKR) is a component of the innate immunity antiviral pathway. PKR is activated upon binding to double-stranded RNA (dsRNA) to undergo dimerization and autophosphorylation. Adenovirus-associated RNA I (VAI) is a short, non-coding transcript whose major function is to inhibit the activity of PKR. VAI contains three domains: an apical stem-loop, a highly structured central domain, and a terminal stem. Previous studies have localized PKR binding to the apical stem and to the central domain. However, the molecular mechanism for inhibition of PKR is not known. We have characterized the stoichiometry and affinity of PKR binding to VAI and several domain constructs using analytical ultracentrifugation and correlated VAI binding and PKR inhibition. Although PKR binding to simple dsRNAs is not regulated by divalent ion, analysis of the interaction of the isolated dsRNA binding domain with VAI reveals that the binding affinity is enhanced by divalent ion. Dissection of VAI into its constituent domains indicates that none of the isolated domains retains the PKR binding affinity or inhibitory potency of the full-length RNA. PKR is capable of binding the isolated terminal stem, but deletion of this domain from VAI does not affect PKR binding or inhibition. These results indicate that both the apical stem and the central domain are required to form a high-affinity PKR binding site. Our data support a model whereby VAI functions as a PKR inhibitor because it binds a monomer tightly but does not facilitate dimerization.