Enzymatic synthesis of cyclic dinucleotide analogs by a promiscuous cyclic-AMP-GMP synthetase and analysis of cyclic dinucleotide responsive riboswitches.

Cyclic dinucleotides are second messenger molecules produced by both prokaryotes and eukaryotes in response to external stimuli. In bacteria, these molecules bind to RNA riboswitches and several protein receptors ultimately leading to phenotypic changes such as biofilm formation, ion transport and secretion of virulence factors. Some cyclic dinucleotide analogs bind differentially to biological receptors and can therefore be used to better understand cyclic dinucleotide mechanisms in vitro and in vivo. However, production of some of these analogs involves lengthy, multistep syntheses. Here, we describe a new, simple method for enzymatic synthesis of several 3', 5' linked cyclic dinucleotide analogs of c-di-GMP, c-di-AMP and c-AMP-GMP using the cyclic-AMP-GMP synthetase, DncV. The enzymatic reaction efficiently produced most cyclic dinucleotide analogs, such as 2'-amino sugar substitutions and phosphorothioate backbone modifications, for all three types of cyclic dinucleotides without the use of protecting groups or organic solvents. We used these novel analogs to explore differences in phosphate backbone and 2'-hydroxyl recognition between GEMM-I and GEMM-Ib riboswitches.

Structural analysis of adenovirus VAI RNA defines the mechanism of inhibition of PKR.

Protein kinase R (PKR) is activated by dsRNA produced during virus replication and plays a major role in the innate immunity response to virus infection. In response, viruses have evolved multiple strategies to evade PKR. Adenovirus virus-associated RNA-I (VAI) is a short, noncoding transcript that functions as an RNA decoy to sequester PKR in an inactive state. VAI consists of an apical stem-loop, a highly structured central domain, and a terminal stem. Chemical probing and mutagenesis demonstrate that the central domain is stabilized by a pseudoknot. A structural model of VAI was obtained from constraints derived from chemical probing and small angle x-ray scattering (SAXS) measurements. VAI adopts a flat, extended conformation with the apical and terminal stems emanating from a protuberance in the center. This model reveals how the apical stem and central domain assemble to produce an extended duplex that is precisely tuned to bind a single PKR monomer with high affinity, thereby inhibiting activation of PKR by viral dsRNA.

Domain interactions in adenovirus VAI RNA mediate high-affinity PKR binding.

Protein kinase R (PKR) is a component of the innate immunity antiviral pathway. PKR is activated upon binding to double-stranded RNA (dsRNA) to undergo dimerization and autophosphorylation. Adenovirus-associated RNA I (VAI) is a short, non-coding transcript whose major function is to inhibit the activity of PKR. VAI contains three domains: an apical stem-loop, a highly structured central domain, and a terminal stem. Previous studies have localized PKR binding to the apical stem and to the central domain. However, the molecular mechanism for inhibition of PKR is not known. We have characterized the stoichiometry and affinity of PKR binding to VAI and several domain constructs using analytical ultracentrifugation and correlated VAI binding and PKR inhibition. Although PKR binding to simple dsRNAs is not regulated by divalent ion, analysis of the interaction of the isolated dsRNA binding domain with VAI reveals that the binding affinity is enhanced by divalent ion. Dissection of VAI into its constituent domains indicates that none of the isolated domains retains the PKR binding affinity or inhibitory potency of the full-length RNA. PKR is capable of binding the isolated terminal stem, but deletion of this domain from VAI does not affect PKR binding or inhibition. These results indicate that both the apical stem and the central domain are required to form a high-affinity PKR binding site. Our data support a model whereby VAI functions as a PKR inhibitor because it binds a monomer tightly but does not facilitate dimerization.

Analysis of PKR-RNA interactions by sedimentation velocity.

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. Some highly structured viral RNAs do not activate and function as PKR inhibitors. In order to define the mechanisms of activation and inhibition of PKR by RNA, it is necessary to characterize the stoichiometries, affinities, and free energy couplings governing the assembly of the relevant complexes. We have found sedimentation velocity analytical ultracentrifugation to be particularly useful in the study of PKR-RNA interactions. Here, we describe protocols for designing and analyzing sedimentation velocity experiments that are generally applicable to studies of protein-nucleic acid interactions. Initially, velocity data obtained at multiple protein:RNA ratios are analyzed using the dc/dt method's to define the association model and to test whether the system is kinetically limited. The sedimentation velocity data obtained at multiple loading concentrations are then globally fitted to this model to determine the relevant association constants. The frictional ratios of the complexes are calculated using the fitted sedimentation coefficients to determine whether the hydrodynamic properties are physically reasonable. We demonstrate the utility of this approach using examples from our studies of PKR interactions with simple dsRNAs, the HIV TAR RNA, and the VAI RNA from adenovirus.

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.