Effects of mutations in stem and loop regions on the structure and function of adenovirus VA RNAI

Abstract

Adenovirus virus-associated (VA) RNAI is required for efficient protein synthesis at late times of adenoviral infection, and in some other situations where double-stranded RNA (dsRNA) is present. It prevents inhibition of protein synthesis by a dsRNA-activated protein kinase and the secondary structure of VA RNAI is though to be important for its activity. To test this idea and to define structures and sequences responsible for VA RNAI activity, we constructed several mutant VA RNA genes and tested them in a transient expression assay. Activity is unaffected by deletions within a small region near the center of the gene, nt 72-85, but it is greatly diminished by deletion or substitution of sequences on the 3' side of this region. The structures of wild-type and mutant RNAs were examined by nuclease-sensitivity analysis. We propose a model for wild-type VA RNAI which differs from that predicted to be the most stable structure. Surprisingly disruption of the longest duplex region in the molecule is tolerated, provided that adjacent structural elements are not rearranged. However, perturbations of elements located in the center of the structure correlate well with loss of function.