RNase L-activating 2'-5' oligoadenylates bind ABCF1, ABCF3 and Decr-1.

A notable signalling mechanism employed by mammalian innate immune signalling pathways uses nucleotide-based second messengers such as 2'3'-cGAMP and 2'-5'-oligoadenylates (OAs), which bind and activate STING and RNase L, respectively. Interestingly, the involvement of nucleotide second messengers to activate antiviral responses is evolutionarily conserved, as evidenced by the identification of an antiviral cGAMP-dependent pathway in Drosophila. Using a mass spectrometry approach, we identified several members of the ABCF family in human, mouse and Drosophila cell lysates as 2'-5' OA-binding proteins, suggesting an evolutionarily conserved function. Biochemical characterization of these interactions demonstrates high-affinity binding of 2'-5' OA to ABCF1, dependent on phosphorylated 2'-5' OA and an intact Walker A/B motif of the ABC cassette of ABCF1. As further support for species-specific interactions with 2'-5' OA, we additionally identified that the metabolic enzyme Decr1 from mouse, but not human or Drosophila cells, forms a high-affinity complex with 2'-5' OA. A 1.4 Å co-crystal structure of the mouse Decr1-2'-5' OA complex explains high-affinity recognition of 2'-5' OA and the mechanism of species specificity. Despite clear evidence of physical interactions, we could not identify profound antiviral functions of ABCF1, ABCF3 or Decr1 or 2'-5' OA-dependent regulation of cellular translation rates, as suggested by the engagement of ABCF proteins. Thus, although the biological consequences of the here identified interactions need to be further studied, our data suggest that 2'-5' OA can serve as a signalling hub to distribute a signal to different recipient proteins.

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.

Molecular basis of CD-NTase nucleotide selection in CBASS anti-phage defense.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are signaling proteins that initiate antiviral immunity in animal cells and cyclic-oligonucleotide-based anti-phage signaling system (CBASS) phage defense in bacteria. Upon phage recognition, bacterial CD-NTases catalyze synthesis of cyclic-oligonucleotide signals, which activate downstream effectors and execute cell death. How CD-NTases control nucleotide selection to specifically induce defense remains poorly defined. Here, we combine structural and nucleotide-analog interference-mapping approaches to identify molecular rules controlling CD-NTase specificity. Structures of the cyclic trinucleotide synthase Enterobacter cloacae CdnD reveal coordinating nucleotide interactions and a possible role for inverted nucleobase positioning during product synthesis. We demonstrate that correct nucleotide selection in the CD-NTase donor pocket results in the formation of a thermostable-protein-nucleotide complex, and we extend our analysis to establish specific patterns governing selectivity for each of the major bacterial CD-NTase clades A-H. Our results explain CD-NTase specificity and enable predictions of nucleotide second-messenger signals within diverse antiviral systems.

STING cyclic dinucleotide sensing originated in bacteria.

Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP-AMP signalling during viral infection and anti-tumour immunity1-5. STING shares no structural homology with other known signalling proteins6-9, which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD+ cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR-STING fusion from the Pacific oyster Crassostrea gigas. Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS-STING pathway in prokaryotic defence against bacteriophages.

Recurrent Loss-of-Function Mutations Reveal Costs to OAS1 Antiviral Activity in Primates.

Immune responses counteract infections but also cause collateral damage to hosts. Oligoadenylate synthetase 1 (OAS1) binds double-stranded RNA from invading viruses and produces 2'-5' linked oligoadenylate (2-5A) to activate ribonuclease L (RNase L), which cleaves RNA to inhibit virus replication. OAS1 can also undergo autoactivation by host RNAs, a potential trade-off to antiviral activity. We investigated functional variation in primate OAS1 as a model for how immune pathways evolve to mitigate costs and observed a surprising frequency of loss-of-function variation. In gorillas, we identified a polymorphism that severely decreases catalytic function, mirroring a common variant in humans that impairs 2-5A synthesis through alternative splicing. OAS1 loss-of-function variation is also common in monkeys, including complete loss of 2-5A synthesis in tamarins. The frequency of loss-of-function alleles suggests that costs associated with OAS1 activation can be so detrimental to host fitness that pathogen-protective effects are repeatedly forfeited.