Magnesium-dependent interaction of PKR with adenovirus VAI.

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Activated PKR phosphorylates the α-subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis in virus-infected cells. Viruses have evolved diverse PKR-inhibitory strategies to evade the antiviral response. Adenovirus encodes virus-associated RNA I (VAI), a highly structured RNA inhibitor that binds PKR but fails to activate. We have characterized the stoichiometry and affinity of PKR binding to define the mechanism of PKR inhibition by VAI. Sedimentation velocity and isothermal titration calorimetry measurements indicate that PKR interactions with VAI are modulated by Mg(2+). Two PKR monomers bind in the absence of Mg(2+), but a single monomer binds in the presence of divalent ion. Known RNA activators of PKR are capable of binding multiple PKR monomers to allow the kinase domains to come into close proximity and thus enhance dimerization. We propose that VAI acts as an inhibitor of PKR because it binds and sequesters a single PKR in the presence of divalent cation.

The PKR-binding domain of adenovirus VA RNAI exists as a mixture of two functionally non-equivalent structures.

VA RNA(I) 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 RNA(I) using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNA(I) 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 RNA(I) conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation.

Systematic deletion of the adenovirus-associated RNAI terminal stem reveals a surprisingly active RNA inhibitor of double-stranded RNA-activated protein kinase.

Adenoviruses use the short noncoding RNA transcript virus-associated (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 (TSDelta21 RNA) efficiently bound PKR and exhibited wild-type inhibitory activity. TSDelta21 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 TSDelta21 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 TSDelta21 RNA bound PKR with wild-type K(d), and overall change in free energy (DeltaG), the thermodynamics of binding (DeltaH and DeltaS) 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.