Foreign double-stranded RNA (dsRNA) generated during the normal course of the viral life cycle serves as a key infection recognition element by proteins of the innate immune response. To circumvent this response, all adenoviruses synthesize at least one highly structured RNA (VA(I)), which, after processing by the RNA silencing machinery, inhibits the innate immune response via a series of interactions with specific protein partners. Surprisingly, VA(I) positively regulates the activity of the interferon-induced 2'-5'-oligoadenylate synthetase (OAS) enzymes, which typically represent a key mechanism whereby host-cell protein translation is attenuated in response to foreign dsRNA. We present data investigating the regulation of the OAS1 isoform by VA(I) derivatives and demonstrate that a processed version of VA(I) lacking the terminal stem behaves as a pseudo-inhibitor of OAS1. A combination of electrophoretic mobility shift assays, dynamic light scattering, and non-denaturing mass spectrometry was used to quantitate binding affinity and characterize OAS1:VA(I) complex stoichiometry. Enzyme assays characterized the ability of VA(I) derivatives to activate OAS1. Finally, the importance of RNA 5'-end phosphorylation state is investigated, and it emphasizes its potential importance in the activation or inhibition of OAS enzymes. Taken together, these data suggest a plausible strategy whereby the virus produces a single RNA transcript capable of inhibiting a variety of members of the innate immune response.