IN-VITRO ANALYSIS OF VIRUS-ASSOCIATED RNA-I (VAI RNA) - INHIBITION OFTHE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN-KINASE PKR BY VAI RNA MUTANTS CORRELATES WITH THE IN-VIVO PHENOTYPE AND THE STRUCTURAL INTEGRITY OF THE CENTRAL DOMAIN

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activatio n of the interferon-induced, double-stranded RNA-activated protein kin ase PKR. The structure of VAI RNA consists of two long, imperfectly ba se-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain, By using a series of adeno virus mutants with linker-scan mutations in the VAI RNA gene, we recen tly showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements o f the central domain are also involved in binding to PKR. In virus-inf ected cells, VAM RNA interacts with latent kinase, which is bound to r ibosomes; this interaction takes place in a complex milieu. To more fu lly understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reprod uced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experiment ally and correlated the structure with function. Without exception, wh en the structure of the short stem-loop of the central domain was pert urbed, the mutants failed to inhibit PKR. Structural disruptions elsew here in the central domain or in the long duplex regions of the molecu le were not deleterious for in vitro function. Thus, these results sup port our previous findings and underscore the importance of the elemen ts present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA invo lves a precise secondary (and tertiary) structure in the central domai n. It has been suggested that VAI RNA does not activate PKR in virus-i nfected cells because of mismatches in the imperfectly base-paired lon g duplex regions. We constructed mutant VAI genes in which the imperfe ctly base-paired duplex regions were converted to perfectly base-paire d regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, whil e they were able to block the activation of PKR better than did the wi ld type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions. Thus, the role of the long duplex regions of VAI RNA is probably to hold th e nucleotide sequences of the central domain in a conformation optimal for function.