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

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, eIF2α, 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 functionin vitroby sequestration of dsRNA. We have utilizedin vitrobinding 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 eIF2α 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 eIF2α and its subsequent phosphorylation.