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 t hat prevents activation of the interferon-induced, dsRNA-activated pro tein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, elF2 alpha, leads to the inhibit ion 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 i n vitro by sequestration of dsRNA We have utilized in vitro binding as says and the yeast two-hybrid system to demonstrate direct interaction s of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1-9 9) located in the regulatory domain and the carboxyl-terminal (amino a cids 367-523) located in the catalytic domain. The amino-terminal regi on of PKR that interacts with pE3 contains a conserved DRBD, suggestin g 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, ds RNA reduces the interaction of pE3 with the carboxyl-terminal region o f PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which elF2 alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may a lso prevent PKR activation by masking the substrate binding domain. Li ke pE3, the amino-terminal region of PKR also interacts with the carbo xyl-terminal domain of PKR. These interactions increase our understand ing of the mechanisms by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed leads us to suggest a novel autoregulat ory mechanism for activation of PKR in which dsRNA binding to the DRBD (s) induces a conformational change that results in release of the ami no terminal region from the substrate binding domain, allowing access to elF2 alpha and its subsequent phosphorylation. (C) 1998 Academic Pr ess