Two basic motifs of reovirus σ3 protein are involved in double-stranded RNA binding

Abstract

It has been reported that the σ3 protein of reovirus can exert an inhibitory effect on the cellular double-stranded RNA (dsRNA) activated protein kinase. Activation of this kinase is thought to be a general mechanism mediating a cellular antiviral response. This enzyme can also be activated upon transfection, resulting in translational inhibition of plasmid-encoded mRNAs. σ3 has an affinity for dsRNA postulated to be responsible for antikinase activity. In the present study, site-directed mutagenesis was performed on two basic regions previously suggested as dsRNA-binding motifs and the mutant σ3 proteins were then expressed in COS cells. These experiments revealed that both motifs are involved in σ3 attachment to RNA. Expression of the mutants lacking RNA-binding capability is stimulated by coexpression of another dsRNA-binding protein, the E3L vaccinia virus protein. These results support a model in which the attachment to dsRNA is directly responsible for the trans-stimulating effect of σ3 on expression of cotransfected genes.Key words: reovirus, PKR, protein synthesis, RNA binding.