Enzyme assays for synthesis and degradation of 2-5As and other 2′-5′ oligonucleotides

Abstract

BackgroundThe 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2′-5′ oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.ResultsHere we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2′-5′ oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD+ as substrates is also presented. The nuclease reactions make use of the isolated 2′-5′ oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2′-5′ oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays.ConclusionsThis paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0043-8) contains supplementary material, which is available to authorized users.

Characteristics of Human OAS1 Isoform Proteins

“…The Pig OAS1 produced few dimers compared to the hOAS1 isomers under the 18 h reaction incubation conditions used. The p42, p44, p46, p48 and p52 hOAS1 proteins were previously shown to have 2-5A synthetase activity [14,21,22]. Consistent with our data, a recent study that directly compared the relative activities of partially purified p42, p44, p46, p48 and p52 expressed in E. coli as maltose-binding protein (MBP) fusion proteins, found that all of the isoforms were able to robustly synthesize 2-5A [23].…”

Activation of the antiviral factor RNase L triggers translation of non-coding mRNA sequences

“…2-5A was synthesized enzymatically in vitro by recombinant human OAS1. First, recombinant OAS1 (p42) containing an N-terminal His tag was expressed in BL21(DE3) E.coli as described before (Poulsen et al, 2015) or with an alternative protocol using autoinduction media (Studier, 2005). Then cells were collected by centrifugation at 7000 g for 15 min at 4˚C.…”

“…To stop the reaction, samples were incubated at 85 ˚C for 15 minutes. Then the samples were filtered (22 µm pore size Millipore filter) and the different 2-5A species were separated on a 16/10 MonoQ column as described before (Poulsen et al, 2015). The same 2-5A fractions from several run was pooled and run again on 16/10 MonoQ column to achieve higher concentrations.…”

“…Activation of RNase L during viral infections begins when OligoAdenylate Synthetases (OASs) bind to and are activated by dsRNA. Once active, OASs synthesize 2-5-oligoadenylate (2-5A) from ATP (Li et al, 2016;Poulsen et al, 2015).…”

Roles of Nicotinamide Adenine Dinucleotide (NAD+) in Biological Systems

“…The assays used dATP and the A(pA)3 tetramer core as substrates. In alternative, a synthetase can use dNTPs together with NAD + as substrates [48]. Therefore, the Ap4A alarmone can be converted also into oligo2-5A.…”