Cyclic dinucleotides (CDNs) play central roles in bacterial homeostasis and virulence as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern recognition receptors in animal cells. Here, we performed a systematic biochemical screen for bacterial signaling nucleotides and discovered a broad family of cGAS / DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize an exceptionally diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analysis of novel signaling nucleotides demonstrate that these molecules activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

Cytosolic DNA triggers innate immune responses through activation of cyclic GMP–AMP synthase (cGAS) and production of the cyclic dinucleotide second messenger 2′3′ cGAMP 1 – 4 . 2′3′ cGAMP is a potent inducer of immune signaling, but no intracellular nucleases are known to cleave 2′3′ cGAMP and prevent activation of the receptor stimulator of interferon genes (STING) 5 – 7 . Through a biochemical screen analyzing 24 mammalian viruses, here we identify poxvirus immune nucleases (poxins) as a family of 2′3′ cGAMP-specific degrading enzymes. Poxins cleave 2′3′ cGAMP to restrict STING-dependent signaling, and deletion of the poxin gene ( B2R ) attenuates vaccinia virus replication in vivo . Crystal structures of vaccinia virus poxin in pre- and post-reactive states define the mechanism of selective 2′3′ cGAMP degradation through metal-independent cleavage of the 3′–5′ bond, converting 2′3′ cGAMP into linear Gp[2′–5′]Ap[3′]. Poxins are conserved in mammalian poxviruses, and remarkably, we further identify functional poxin homologues in the genomes of moths and butterflies and the baculoviruses which infect them. Baculovirus and insect host poxin homologues retain selective 2′3′ cGAMP degradation activity, suggesting an ancient role for poxins in cGAS-STING regulation. Our results define poxins as a family of 2′3′ cGAMP-specific nucleases and demonstrate a mechanism for how viruses evade innate immunity.

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.