Adenoviruses use the short noncoding RNA transcript virusassociated (VA) RNA I 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 RNA I 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 (TS⌬21 RNA) efficiently bound PKR and exhibited wild-type inhibitory activity. TS⌬21 RNA stability was strongly influenced by solution pH, indicating the involvement of a protonated base within the VA RNA I central domain tertiary structure. Gel filtration chromatography and isothermal titration calorimetry analysis indicated that wild-type VA RNA I and TS⌬21 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 TS⌬21 RNA bound PKR with wild-type K d , and overall change in free energy (⌬G), the thermodynamics of binding (⌬H and ⌬S) were significantly altered. These results demonstrate that the VA RNA I terminal stem is entirely dispensable for inhibition of PKR. Potentially, VA RNA I is therefore a truly bi-functional RNA; Dicer processing of the VA RNA I terminal stem saturates the RNA interference system while generating a "mini-VA RNA I " molecule that remains fully active against PKR.The interferon-induced double-stranded RNA (dsRNA) 4 -activated protein kinase (PKR) is a key component of the innate immune response that forms the first line of intracellular defense against viral infection (1, 2). PKR regulates translation initiation by phosphorylating the eukaryotic initiation factor 2 (eIF2) ␣-subunit at serine 51. The large increase in affinity of the phosphorylated form for its guanosine exchange factor (eIF2B) results in competitive inhibition and the reduction in available eIF2⅐GTP⅐Met-tRNA i Met ternary complex leads to a sharp reduction in both cellular and viral protein expression (3-5). Viruses devote large portions of their genomes to evading such host defenses and have evolved many different strategies to counter the PKR-mediated response (6). For example, Epstein-Barr virus and adenovirus produce large quantities of short noncoding RNA transcripts, EBER (7, 8) and VA RNAs, respectively (9, 10), that bind directly to PKR but inhibit rather than activate the kinase activity.All adenoviruses encode at least one VA RNA sequence (VA RNA I ) of ϳ160 nucleotides that is transcribed by the host RNA polymerase III and accumulates to very high concentrations in the late stages of infection (11, 12). Although VA RNA I sequences from different virus serotypes vary considerably, all can be drawn in a similar extended structure consisting of three major ...

Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNAI were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNAI was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent Tm of ∼60°C. In contrast, the Apical Stem unfolds independently and with much higher apparent Tm of ∼83°C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNAI Central Domain structure essential for PKR inhibitory activity was identified.

VA RNAI is a non-coding adenoviral transcript that counteracts the host cell anti-viral defenses such as immune responses mediated via PKR. We investigated potential alternate secondary structure conformations within the PKR-binding domain of VA RNAI using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNAI apical stem can adopt two different conformations and that it exists as a mixed population of these two structures. In contrast, in two sequence variants we designed to eliminate one of the possible structures, while leaving the other intact, each formed a unique secondary structure. This clarification of the apical stem pairing also suggests a small alteration to the apical stem–loop secondary structure. The relative ability of the two apical stem conformations to bind PKR and inhibit kinase activity was measured by isothermal titration calorimetry and PKR autophosphorylation inhibition assay. We found that the two sequence variants displayed markedly different activities, with one being a significantly poorer binder and inhibitor of PKR. Whether the presence of the VA RNAI conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation.

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn 2؉ cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.Since the discovery of protein regulation by RNA interference (RNAi), RNA, as both a target and effector molecule has been widely researched for therapeutic purposes (1, 2). To the original exogenous small interfering RNAs (siRNA), microRNA, non-coding RNA, and long non-coding RNA have been added; all of which are capable of fine regulation of their target protein(s), and thereby cellular processes (3). This has opened up the possibility of managing both genetic and acquired diseases by modifying the levels of the important disease-associated proteins or essential pathogen-associated proteins using RNA-based technologies (4, 5). Moreover, RNA has the ability to fold into complex tertiary structures that rival antibodies in their potential diversity (provided by the sequence context of the RNA)(6). This conformational heterogeneity makes RNA an ideal effector molecule to bind to and inactivate proteins in a structure-specific manner, similar to the antibody-antigen interaction. Such RNA molecules are called aptamers and have been used both in the laboratory, e.g. to distinguish diseased from wild-type prion protein conformations (7), and in the clinic, e.g. to treat age-related macular degeneration (Macugen). Additionally, our laboratory has recently been developing a clinically relevant RNA-based aptamer to prevent HIV-1 infection (8, 9).The "RNA world" hypothesis states that life originated using RNA as the inherited genetic molecule, which was superseded by DNA due to...