Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.

摘要: Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred as p68 on basis its Mr 68,000 in human cells). Viruses must exert this control avoid extensive phosphorylation alpha subunit eukaryotic initiation factor 2 (eIF-2) by and resultant negative effects synthesis initiation. To begin define molecular mechanisms underlying regulation, we optimized expression an vitro transcription-translation system utilizing full-length cDNA clone. The vitro-expressed was autophosphorylated response dsRNAs heparin a manner similar that for native provided inhibitor, 2-aminopurine, present during translation reaction. Further, activated efficiently phosphorylated natural substrate, eIF-2. Binding experiments revealed expressed complexed with dsRNA activator, reovirus dsRNA, well adenovirus-encoded VAI RNA. Interestingly, both RNAs RNA also molecules lacked carboxyl terminus all catalytic domains. Deletion analysis confirmed amino contained critical determinants binding. bound to, but failed activate, containing single acid substitution invariant lysine 295 domain II. Taken together, these data demonstrate permits detailed mutagenic regions required interaction virus-encoded activators repressors.