Extended Data Fig. 5: cUMP–AMP recognition helps to define innate immune receptor specificity.

a, e, Quantification of nucleotide interactions with the host receptors STING (a) or RECON (e), measured using a radiolabelled nucleotide bound to a concentration gradient of host protein, separated in a native PAGE gel shift (0, 4, 20, 100 μM protein). Quantification of the gels shown in b and f. Data are representative of n = 2 independent experiments. b, f, Native PAGE gel shift analysis of STING (b) or RECON (f) complex formation with indicated radiolabelled CDNs. Proteins are titrated at 0 (−), 4, 20 and 100 μM. STING readily binds all cyclic dipurine species, but does not form a high-affinity complex with cUMP–AMP. RECON readily binds all 3′,3′-CDN species that contain at least one adenine base, including cUMP–AMP. Data are representative of two independent experiments. c, In-cell STING reporter assay. Induction of an IFNβ reporter in HEK293T cells transfected with a concentration gradient of plasmid-overexpressing enzymes as indicated. DncV and CdnE were expressed with N-terminal MBP tags and IFNβ reporter induction was compared as fold over empty vector, shown as (−). Data are mean ± s.e.m. for n = 3 technical replicates and are representative of two independent experiments. d, Western blot of MBP-tagged DncV and CdnE expressed from plasmids analysed in c to validate in vivo expression. Data are representative of two independent experiments. Gel source data are available in Supplementary Fig. 1. g, Gel shift analysis as in f, with protein titration to measure the relative affinity of the RECON–cUMP–AMP interaction. Protein concentrations listed below. Data are representative of 2 independent experiments.