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

Abstract

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 ...

Induction of type I interferon by adenovirus-encoded small RNAs

“…*P < 0.01. 2α via dsRNA-induced protein kinase R activation (19)(20)(21). However, the roles played by VA-RNAs in the Ad vector-induced innate immune response have not been elucidated.…”

“…VA-RNAs are synthesized by RNA polymerase III, are ≈160 nucleotides long, and are structurally highly conserved. Previous studies have demonstrated that VARNAs induce the phosphorylation of eukaryotic initiation factor-2α via dsRNA-induced protein kinase R activation (19)(20)(21).…”

Dissection of the Adenoviral VA RNAI Central Domain Structure Reveals Minimum Requirements for RNA-mediated Inhibition of PKR

“…1A, nucleotides in outline font) (11,12,21). We have shown that the stability of the central domain structure is also highly sensitive to solution pH, suggesting the involvement of a protonated base-base tertiary interaction (8). Finally, Mg 2ϩ and PKR have been shown to induce similar structural rearrangements in the central domain (14), and Mg 2ϩ appears to fine-tune the mode and affinity of the PKR-VA RNA I interaction (22).…”

“…Specific regulation of host genes by VA RNA Iderived microRNAs has been proposed (5,6), but a precise functional role in adenoviral replication has not yet been established (7). The remaining portion of the Dicer-processed VA RNA I is a truncated form of the transcript, lacking the viral microRNA sequences at both the 5Ј and 3Ј ends, that retains full activity against PKR (8). Paradoxically, full-length VA RNA I activates a second protein of the innate immune response, 2Ј-5Ј-oligoadenylate synthetase 1 (OAS1), leading to downstream activation of RNaseL and degradation of viral and cellular RNAs (9).…”

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

“…Elucidating the mechanism by which PKR function is subverted by viral products has enhanced our understanding of how PKR and other eIF2α family kinases naturally function. In particular, viral mimicry appeared as a recurring theme: analysis of the pseudosubstrate inhibitor K3L from vaccinia virus revealed key molecular determinants required for PKR recognition of its natural substrate eIF2α (10)(11)(12); analysis of the RNA binding protein E3L from vaccinia virus revealed how PKR itself senses dsRNA through comparable infrastructure (13)(14)(15); and analysis of the RNA inhibitor VAI from adenovirus showed how a structured RNA decoy can competitively engage the dsRNA-binding domains of PKR in a manner that circumvents kinase domain activation (16)(17)(18).…”