The binding site of the RNA-dependent protein kinase (PKR) on EBER1 RNA from Epstein-Barr virus

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the eukaryotic initiation factor 2alpha, rendering the translation machinery inactive. Viruses have developed strategies for preventing the action of PKR, one of which is the production of small RNAs that inhibit the enzyme. Epstein-Barr virus (EBV) encodes EBER1, a 167 nucleotide non-coding RNA that is constitutively expressed by the EBV-infected cells. EBER1 binds PKR in vitro and has been shown to prevent inhibition of translation by PKR in vitro. We used affinity cleavage by the EDTA-Fe-modified double-stranded RNA-binding domain (dsRBD) of PKR to show that stem-loop IV (nucleoticles 87-123) of EBER1 makes specific contacts with the dsRBD. To further demonstrate the specificity of this interaction, we generated a deletion mutant of EBER1, comprising only stem-loop IV (mEBER1). Cleavage patterns produced on mEBER1 by the bound dsRBD were remarkably similar to those found on full-length EBER1. Using cleavage data from two different dsRBD mutants, we present a model of the interaction of PKR dsRBD and mEBER1.