Adenovirus VA RNAs (virus-associated RNAs) are small polymerase III transcripts that are required for efficient initiation of mRNA translation late in adenovirus infection. VAI RNA prevents double-stranded RNA (dsRNA) activation of the interferon-induced protein kinase (DAI kinase). Activation of this kinase results in phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) and correlates with inhibition of translation initiation. In this report we show growth complementation of adenoviruses harboring deletions in the VAI gene in cell lines expressing a serine-to-alanine mutant of eIF-2 alpha. This serine-to-alanine mutant is resistant to phosphorylation by DAI kinase. These results directly show that the primary function of VAI RNA in the lytic adenovirus infection is the inhibition of eIF-2 alpha phosphorylation by DAI kinase and identify eIF-2 alpha as the target that mediates the effects of DAI kinase activation. Cells that express a mutant eIF-2 alpha will enable the isolation of specific host-range mutants for other types of viruses that are defective in the ability to inhibit DAI kinase.