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.

Systematic Deletion of the Adenovirus-associated RNAI Terminal Stem Reveals a Surprisingly Active RNA Inhibitor of Double-stranded RNA-activated Protein Kinase

“…In contrast, the pH dependence is reversed for bp RNA, which lacks the (A⅐C) ϩ mismatch pair (Fig. 1). primary binding site and its complex central domain as the major determinant of inhibitory activity (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Although far less extensively examined, prior mutagenesis suggested that an intact terminal stem structure, particularly adjacent to the central domain, might also contribute to inhibition of PKR (21).…”

“…Although VA RNA I sequences from different virus serotypes vary considerably, all can be drawn in a similar extended structure consisting of three major domains (13,14) as follows: the terminal stem (including the paired 5Ј and 3Ј ends), a central domain, and the apical stem capped by a loop structure (Fig. 1).…”

“…1). The apical stem and central domain are responsible for binding and inhibition of PKR, respectively, and the structural requirements for these roles have been thoroughly dissected (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Remarkably, this functional division (22) is mirrored by a structural division between the apical stem and the central domain, which are essentially independent domains within the VA RNA I global architecture (25).…”

Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

“…Although the nucleotide sequences of VA RNAs are poorly conserved between adenovirus serotypes, their secondary structure has been well maintained and can generally be divided into a terminal stem, a panhandle apical stem, and a more structured central domain ( Fig. 1) (15,32,36). Mutagenic studies indicate that the apical stem-loop is required for binding to PKR and that the central domain is involved in the inhibition of PKR activation (35)(36)(37).…”

“…1) (15,32,36). Mutagenic studies indicate that the apical stem-loop is required for binding to PKR and that the central domain is involved in the inhibition of PKR activation (35)(36)(37). The terminal stem is essential for binding to Exp 5, which mediates VA transport to the cytoplasm (16,17).…”