Abstract
Adenoviruses use the short noncoding RNA transcript virus-associated (VA) RNAI to counteract two critical elements of the host cell defense system, innate cellular immunity and RNA interference, mediated by the double-stranded RNA-activated protein kinase (PKR) and Dicer/RNA-induced silencing complex, respectively. We progressively shortened the VA RNAI terminal stem to examine its necessity for inhibition of PKR. Each deletion, up to 15 bp into the terminal stem, resulted in a cumulative decrease in PKR inhibitory activity. Remarkably, however, despite significant apparent destabilization of the RNA structure, the final RNA mutant that lacked the entire terminal stem (TSΔ21 RNA) efficiently bound PKR and exhibited wild-type inhibitory activity. TSΔ21 RNA stability was strongly influenced by solution pH, indicating the involvement of a protonated base within the VA RNAI central domain tertiary structure. Gel filtration chromatography and isothermal titration calorimetry analysis indicated that wild-type VA RNAI and TSΔ21 RNA form similar 1:1 complexes with PKR but that the latter lacks secondary binding site(s) that might be provided by the terminal stem. Although TSΔ21 RNA bound PKR with wild-type Kd, and overall change in free energy (ΔG), the thermodynamics of binding (ΔH and ΔS) were significantly altered. These results demonstrate that the VA RNAI terminal stem is entirely dispensable for inhibition of PKR. Potentially, VA RNAI is therefore a truly bi-functional RNA; Dicer processing of the VA RNAI terminal stem saturates the RNA interference system while generating a “mini-VA RNAI” molecule that remains fully active against PKR.
Highlights
JUNE 20, 2008 VOLUME 283 NUMBER 25 immune response that forms the first line of intracellular defense against viral infection [1, 2]
VA RNAI 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, a central domain, and the apical stem capped by a loop structure (Fig. 1)
The end of the stem can be altered without reducing RNA function [13], it is possible that an intact terminal stem may be required to act as a virus-associated RNAI; TS, (VA RNAI) terminal stem; T7 RNAP, T7 RNA polymerase; Isothermal Titration Calorimetry (ITC), isothermal titration calorimetry; nt, nucleotide; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid
Summary
Mutagenesis and Preparation of RNA in Vitro Transcripts— A plasmid encoding adenovirus type 2 (Ad2) VA RNAI was created with a 5Ј-T7 RNA polymerase (T7 RNAP) promoter and 3Ј-hepatitis delta virus ribozyme sequence followed by a DraI restriction site for run-off transcription, as described previously [27]. The VA RNAI terminal stem (TS) was first deleted in three successive segments of 5 bp each to create the mutants TS⌬5, TS⌬10, and TS⌬15 For these RNAs, the 3 terminal bp of the wild-type sequence were maintained to provide a strong promoter for T7 RNA polymerase (T7 RNAP). Up to five melting curves were collected in each experiment with the sixth cell containing only buffer and fitted with an in-sample temperature probe. Wild-type VA RNAI and each terminal stem deletion mutant RNA were diluted into the same buffer to generate a 5ϫ stock for each point in the inhibition assay (see Fig. 3 legend for details). Control experiments without poly(I)1⁄7poly(C)-activating RNA showed that none of the VA RNAI samples activated PKR. The PKR concentration was 3 M for all experiments that contained protein, whereas the RNA concentration was 0.75 M (4:1 protein to RNA ratio), 1.5 M (2:1), 3 M (RNA alone and 1:1), or 6 M (1:2)
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