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

Abstract

The RNA-dependent protein kinase (PKR) is an interferoninduced, RNA-activated enzyme that phosphorylates the eukaryotic initiation factor 2α, 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 (nucleotides 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.

The Epstein-Barr Virus BILF1 Gene Encodes a G Protein-Coupled Receptor That Inhibits Phosphorylation of RNA-Dependent Protein Kinase

“…Until now, alteration of PKR phosphorylation was only reported for cellular non-GPCRrelated signal transduction pathways, such as those triggered by viral double-stranded RNA, lipopolysaccharide, interleukin-1, tumor necrosis factor alpha, heat shock, platelet-derived growth factor, or interleukin-3 deprivation (reviewed in reference 52). Some EBV-specific factors, such as the RNA molecules EBER1 and EBER3 (50), as well as the lytic-phase posttranslational regulator protein SM (43) were also shown to reduce PKR phosphorylation. Thus, the discovery of EBV GPCR-dependent PKR inhibition sheds new light on the PKRmediated cellular response to biological stress.…”

Protection from Interferon-Induced Apoptosis by Epstein-Barr Virus Small RNAs Is Not Mediated by Inhibition of PKR

“…Within reticulocyte lysates, the EBERs, like the structurally comparable adenovirus VAI small RNA, bind PKR and inhibit its repression of translation in a dose-dependent manner (3,34). Concordantly, stem-loop IV of EBER-1 is bound in a specific manner by PKR in vitro through its dsRBM I, but not dsRBM II (42), a feature apparently distinct from activating dsRNAs that interact with both sites, presumably relieving an autoinhibitory influence of dsRBM II on the PKR catalytic domain (36). The EBER RNAs, furthermore, are able to rescue replication of adenoviruses that lack the VAI RNA gene (2), suggesting that the EBERs share with the VAI RNA the ability to inhibit PKR in vivo.…”

The Challenge of Viral snRNPs

“…Like most (Fok et al 2006b). PKR binding at stem-loop IV has been proposed by Vuyisich et al (2002) and McKenna et al (2006). (B) Lack of EBER nucleocytoplasmic shuttling.…”

“…The EBERs were initially proposed to function like adenovirus VA1 RNA by inactivating the dsRNA-dependent protein kinase, PKR (Mathews and Shenk 1991), because they partially support lytic growth of a mutant adenovirus deleted for VA1 (Bhat and Thimmappaya 1983) and bind PKR in vitro (Sharp et al 1993;Vuyisich et al 2002;McKenna et al 2006). However, since EBERs appear to be nuclear (Howe and Steitz 1986;Barletta et al 1993) whereas PKR is largely cytoplasmic , it seemed unlikely that EBERs could combat this host antiviral defense by inhibiting PKR if they truly reside in a different cellular compartment.…”