EFFECT OF SINGLE-BASE SUBSTITUTIONS IN THE CENTRAL DOMAIN OF VIRUS-ASSOCIATED RNA-I ON ITS FUNCTION

Adenoviruses use virus associated RNA I (VAI RNA) to counteract the ce llular antiviral response mediated by the interferon-induced, double-s tranded-RNA-activated protein kinase PKR. VAI RNA is a highly structur ed small RNA which consists of two long duplex regions connected at th e center by a complex, short stem-loop. This short stem-loop and the a djacent base-paired regions, referred to as the central domain, bind t o PKR and inactivate it. Currently it is not known whether binding of VAI RNA to PKR is dependent solely on the secondary (and tertiary) str ucture of the central domain or whether nucleotide sequences in the ce ntral domain are also critical for this interaction. To address this q uestion, 54 VAI mutants with single-base substitution mutations in the central domain of the RNA were constructed, and their capacities to i nhibit the autophosphoryation of PKR in vitro were determined. It was found that although about half of the mutants inhibited PKR activity a s efficiently as the wild type, a significant number of mutants lost t he inhibitory activity substantially, without a perceptible change in their secondary structures. These results indicate that, in addition t o secondary structure, at least some nucleotides in the central domain may be critical for the efficient function of VAI RNA.