Recombinant vaccinia virus K3L gene product prevents activation of double- stranded RNA-dependent, initiation factor 2α-specific protein kinase

Deletion of the vaccinia virus K3L gene, a homologue of the α subunit of protein synthesis initiation factor 2, has been reported to reduce the ability of the virus to grow in interferon-treated cells (Beattie, E., Tattaglia, J., and Paoletti, E. (1991) Virology 183, 419-422). Purified recombinant K3L gene product, pK3(r), has potent effects on activation of double-stranded (ds) RNA-dependent, initiation factor-2α (eIF-2α)-specific protein kinase (PKR) in in vitro reactions. Recombinant pK3 prevents the inhibition of protein synthesis by dsRNA in a cell-free translation system from rabbit reticulocytes at levels equal to, or lower than, the level of endogenous eIF-2α. In the cell-free translation system, pK3(r) exerts its effects at all dsRNA concentrations tested, by preventing phosphorylation of eIF-2α. In addition, pK3(r) reduces the autophosphorylation of immunopurified PKR, as well as its ability to phosphorylate the α subunit of purified eIF-2. At 400 mM NaCl, in vitro translated [³⁵S]methionine- radiolabeled pK3 can be co-immunoprecipitated with human PKR, using a monoclonal antibody to PKR. This tight binding is consistent with a role for pK3 as a pseudosubstrate for the kinase, and identifies the amino-terminal 30% of eIF-2α as the domain recognized by the eIF-2α-specific protein kinases. In addition, the tight binding opens up the possibility of using binding assays to identify functional domains within the kinase and pK3. Recombinant pK3 also prevents activation of the heme-sensitive eIF-2α- specific protein kinase, eIF-2α-PK(h), in both cell-free translation systems as well as in partially purified preparations. This suggests some similarity between the eIF-2α binding domains of the two eIF-2α specific protein kinases.