The Vaccinia Virus E3L Gene Product Interacts with both the Regulatory and the Substrate Binding Regions of PKR: Implications for PKR Autoregulation

Abstract

The vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that prevents activation of the interferon-induced, dsRNA-activated protein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, eIF2alpha, leads to the inhibition of protein synthesis, a process involved in defense against virus infection. The E3L gene product has a conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has been shown to function in vitro by sequestration of dsRNA. We have utilized in vitro binding assays and the yeast two-hybrid system to demonstrate direct interactions of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1-99) located in the regulatory domain and the carboxyl-terminal (amino acids 367-523) located in the catalytic domain. The amino-terminal region of PKR that interacts with pE3 contains a conserved DRBD, suggesting that PKR can form nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA binding proteins. Interaction of pE3 with the amino-terminal region of PKR is enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the carboxyl-terminal region of PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which eIF2alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may also prevent PKR activation by masking the substrate binding domain. Like pE3, the amino-terminal region of PKR also interacts with the carboxyl-terminal domain of PKR. These interactions increase our understanding of the mechanisms by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed leads us to suggest a novel autoregulatory mechanism for activation of PKR in which dsRNA binding to the DRBD(s) induces a conformational change that results in release of the amino terminal region from the substrate binding domain, allowing access to eIF2alpha and its subsequent phosphorylation.

Genome-wide analysis of vaccinia virus protein–protein interactions

“…We detected the selfassociation of E3L, a double-stranded RNA-binding protein involved in IFN resistance (52). This interaction had been previously described, as well as that between E3L and the substrate binding domain of the host IFN-induced doublestranded RNA-activated protein kinase (53,54). An interaction between the K1L protein, required for replication in rabbit kidney and human cell lines (55), with the uncharacterized C10L protein was recently found by using another version of the yeast two-hybrid system (A. Grunhaus and B.M., unpublished data) and was confirmed in the present screening.…”

The Ebola virus VP35 protein functions as a type I IFN antagonist

“…These inhibitory functions may be mediated by the binding of dsRNA by NS1 (21,23,52). The vaccinia virus E3L protein also blocks the activation of both PKR (37)(38)(39)(40) and OAS (41). Further, E3L expression promotes growth of vaccinia virus in the presence of IFN (40,42).…”

IRF3 and IRF7 Phosphorylation in Virus-infected Cells Does Not Require Double-stranded RNA-dependent Protein Kinase R or IκB Kinase but Is Blocked by Vaccinia Virus E3L Protein

“…However, sequestering of dsRNA alone is probably insufficient to inhibit IRF phosphorylation since other RNA-binding proteins were unable to mimic this action of E3L. Interestingly, the non-RNA binding amino terminus of E3L is required for full inhibition of PKR (63,64) and must interact with PKR, not simply sequester its activator dsRNA to block catalytic activity. The amino terminus of E3L also induces multimerization (69) that may be necessary for its function.…”

“…E3L binds dsRNA and has been thought of as a competitive inhibitor of PKR that sequesters dsRNA (56,60) and directly inhibits PKR through protein-protein interaction following binding to dsRNA (63,64). We tested the requirement of dsRNA binding for the inhibition of IRF7 phosphorylation by E3L using a point mutant version of E3L, in which lysine 167 was converted to alanine (K167A), and is incapable of binding dsRNA (65).…”