Uncoupling of RNA binding and PKR kinase activation by viral inhibitor RNAs.
Protein kinase RNA-activated (PKR) is a serine/threonine kinase that contains an N-terminal RNA-binding domain and a C-terminal kinase domain. Upon binding double-stranded RNA (dsRNA), PKR can become activated and phosphorylate cellular targets, such as eukaryotic translation initiation factor 2alpha (eIF-2alpha). Phosphorylation of eIF-2alpha results in attenuation of protein translation by the ribosome in either a general or an mRNA-specific manner. Therefore, the interaction between PKR and dsRNAs represents a crucial host cell defense mechanism against viral infection. Viruses can circumvent PKR function by transcription of virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We present here a biophysical characterization of the interactions between human PKR and two viral inhibitor RNAs, EBER(I) (from Epstein-Barr virus) and VA(I) (from human adenovirus). Autophosphorylation assays confirmed that both EBER(I) and VA(I) are inhibitors of PKR activation, and profiled the kinetics of the inhibition. Binding affinities of dsRNAs to PKR double-stranded RNA-binding domains (dsRBDs) were determined by isothermal titration calorimetry and gel electrophoresis. A single stem-loop domain from each inhibitory RNA mediates the interaction with both dsRBDs of PKR. The binding sites on inhibitor RNAs and the dsRBDs of PKR have been mapped by NMR chemical shift perturbation experiments, which indicate that inhibitors of PKR employ similar surfaces of interaction as activators. Finally, we show that dsRNA binding and inactivation are non-equivalent; regions other than the dsRBD stem-loops of inhibitory RNA are required for inhibition.
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
Sean A
SA
McKenna
Insil
I
Kim
Corey W
CW
Liu
Joseph D
JD
Puglisi
D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D004789:Enzyme Activation; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C099537:Epstein-Barr virus encoded RNA 1; D016337:RNA, Catalytic; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D011994:Recombinant Proteins; C419304:VA1-ribozyme chimera; D019892:eIF-2 Kinase