The virus-induced cyclic dinucleotide 2'3'-c-di-GMP mediates STING-dependent antiviral immunity in Drosophila.

In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP, which triggers STING-dependent immunity. In Drosophila melanogaster, two cGAS-like receptors (cGLRs) produce 3'2'-cGAMP and 2'3'-cGAMP to activate STING. We explored CDN-mediated immunity in 14 Drosophila species covering 50 million years of evolution and found that 2'3'-cGAMP and 3'2'-cGAMP failed to control infection by Drosophila C virus in D. serrata and two other species. We discovered diverse CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including 2'3'-c-di-GMP. This CDN was a more potent STING agonist than cGAMP in D. melanogaster and it also activated a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for understanding the function and regulation of this emerging family of pattern recognition receptors in animal innate immunity.

cGLRs are a diverse family of pattern recognition receptors in innate immunity.

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

CBASS to cGAS-STING: The Origins and Mechanisms of Nucleotide Second Messenger Immune Signaling.

Host defense against viral pathogens is an essential function for all living organisms. In cell-intrinsic innate immunity, dedicated sensor proteins recognize molecular signatures of infection and communicate to downstream adaptor or effector proteins to activate immune defense. Remarkably, recent evidence demonstrates that much of the core machinery of innate immunity is shared across eukaryotic and prokaryotic domains of life. Here, we review a pioneering example of evolutionary conservation in innate immunity: the animal cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) signaling pathway and its ancestor in bacteria, CBASS (cyclic nucleotide-based antiphage signaling system) antiphage defense. We discuss the unique mechanism by which animal cGLRs (cGAS-like receptors) and bacterial CD-NTases (cGAS/dinucleotide-cyclase in Vibrio (DncV)-like nucleotidyltransferases) in these pathways link pathogen detection with immune activation using nucleotide second messenger signals. Comparing the biochemical, structural, and mechanistic details of cGAS-STING, cGLR signaling, and CBASS, we highlight emerging questions in the field and examine evolutionary pressures that may have shaped the origins of nucleotide second messenger signaling in antiviral defense.

cGLRs are a diverse family of pattern recognition receptors in animal innate immunity.

cGAS (cyclic GMP-AMP synthase) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2'3'-cGAMP that activates the protein STING and downstream immunity. Here we discover cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in animal innate immunity. Building on recent analysis in Drosophila , we use a bioinformatic approach to identify >3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screen of 140 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of alternative nucleotide signals including isomers of cGAMP and cUMP-AMP. Using structural biology, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Together our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity.

Species-specific self-DNA detection mechanisms by mammalian cyclic GMP-AMP synthases.

The mechanisms by which innate immune receptors mediate self-nonself discrimination are unclear. In this study, we found species-specific molecular determinants of self-DNA reactivity by cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). Human cGAS contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream amino (N)-terminal domain. The cGAS proteins from several nonhuman primate species exhibited a similar pattern of self-DNA reactivity in cells, but chimpanzee cGAS was inactive even when its amino-terminal domain was deleted. In contrast, the N terminus of mouse cGAS promoted self-DNA reactivity. When expressed within tumors, only self-DNA-reactive cGAS proteins protected mice from tumor-induced lethality. In vitro studies of DNA- or chromatin-induced cGAS activation did not reveal species-specific activities that correlate with self-DNA reactivity observed in macrophages. Cell biological analysis revealed that self-DNA reactivity by human cGAS, but not mouse cGAS, correlated with localization to mitochondria. We found that epitope tag positions affected self-DNA reactivity in cells and that DNA present in cell lysates undermines the reliability of cGAS biochemical fractionations. These studies reveal species-specific diversity of cGAS functions, even within the primate lineage, and highlight experimental considerations for the study of this innate immune receptor.

cGAS-like receptors sense RNA and control 3'2'-cGAMP signalling in Drosophila.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that produces the second messenger cG[2'-5']pA[3'-5']p (2'3'-cGAMP) and controls activation of innate immunity in mammalian cells1-5. Animal genomes typically encode multiple proteins with predicted homology to cGAS6-10, but the function of these uncharacterized enzymes is unknown. Here we show that cGAS-like receptors (cGLRs) are innate immune sensors that are capable of recognizing divergent molecular patterns and catalysing synthesis of distinct nucleotide second messenger signals. Crystal structures of human and insect cGLRs reveal a nucleotidyltransferase signalling core shared with cGAS and a diversified primary ligand-binding surface modified with notable insertions and deletions. We demonstrate that surface remodelling of cGLRs enables altered ligand specificity and used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in the model organism Drosophila melanogaster. We show that RNA recognition activates Drosophila cGLR1 to synthesize the novel product cG[3'-5']pA[2'-5']p (3'2'-cGAMP). A crystal structure of Drosophila stimulator of interferon genes (dSTING) in complex with 3'2'-cGAMP explains selective isomer recognition, and 3'2'-cGAMP induces an enhanced antiviral state in vivo that protects from viral infection. Similar to radiation of Toll-like receptors in pathogen immunity, our results establish cGLRs as a diverse family of metazoan pattern recognition receptors.